Design of Target Support Columns Using EPS Foam

集团总部与所辖公司管理权限划分细则集团总部与所辖公司的管理权限划分细则如下：财务管理方面，全资开发公司负责付款、报销、借款等事项，并备案审批其他事项。

同时，全资开发公司还需要管理财务人员、财务资金、计划及融资等日常事务，以及制定年度、季度、月度财务预算计划。

此外，全资开发公司还需要提供财务情况、销售情况、资金情况、财务人员工作情况、资金计划等信息，并制定财务管理工作及相关人员的考核办法、薪酬、福利、培训管理办法。

全资子公司的资金调配、公司融资管理、财务管理中的各项基础资料、报表及财务报告，以及财务部经理及以上人员的任免也归全资开发公司负责。

而财务部经理级以下人员的聘用、解聘、调配以及人员配置，则需要经过审核和审批。

应收账款明细调减、对没有投资关系的企业提供贷款担保、各类型的对外融资、对外单位担保、开立新的银行账户、核算制度、核算口径的变化、启用新的会计套账、财务管理实施细则的制定、修改、各种重大投资计划等事项需要经过审核、审批或决策。

对于单笔交际费用在2-8万元以内的管理费用，以及单笔开发成本、销售费用等在150万元以上的事项，需要审核和备案。

单笔交际费用在8万元以上的管理费用，以及除交际费用以外的管理费用单笔在10万元以上的事项，则需要经过审批。

应收账款管理方面，除交际费用以外的管理费用单笔在15-20万元以内的事项需要审核，单笔交际费用在2-8万元以内的管理费用需要备案。

除交际费用以外的管理费用单笔在20万元以上的事项，以及总经理发生的费用，需要经过审批。

办公用品《物资采购申请单》单笔在1.5-3万元的事项需要备案。

办公用品采购申请单，单笔金额在3-5万元之间。

办公用品采购申请单，单笔金额在5万元以上。

员工因公现金借款单笔金额在8-10万元之间。

员工因公现金借款单笔金额在10万元以上。

员工因公借款超出其级别总额限制。

员工借款需延期归还。

特殊情况下的对外借款和员工因私借款。

开发成本、销售费用等单笔在600万元以上。

本月，刚刚登上世界最高建筑新科状元宝座的迪拜哈利法塔盛大揭幕但建筑师们不会就此止步。

世界第一总是有无穷的魔力，吸引着人们去征服更高的高度，下面的这八个建筑项目都计划打破记录，成为新任的世界最高建筑。

By : Adam HadhazyPublished: January 21, 2010 11:21 PMFrom: Popular MechanicsWeb site: /technology/engineering/architecture/4343115Top 8 Skyscrapers That Will Push the Limits of DesignThis month, the Burj Khalifa in Dubai climbed higher than any other previous structure ever built. But architects won't rest there. Here are eight building plans trying to capture the title as the next tallest tower.The Burj Khalifa, currently the tallest tower in the world, officially opened in Dubai on Jan. 4 amid an impressive pyrotechnics display that highlighted the tower's 2716.5-feet of aluminum and steel, and its 26,000 hand-cut glass panels. The Burj Khalifa blows away the next-nearest skyscraper, which is Taiwan's 1670-foot Taipei 101, and the building has even surpassed ultra-tall, ground-cable-supported radio antennas.Architects' vertical leapfrogging, however, isn't likely to stop at the Burj Khalifa. While the tower will be a tough one to beat, it is likely to remain at the pinnacle for only about another half-dozen years. Developers around the world have proposed numerous new skyscrapers. Some projects have leapt off the drawing boards, though plans for many record-breaking towers have been scuttled because of the global economic spasms of the past couple years. (The original name of the Burj Khalifa, the Burj Dubai, was changed at the last minute to recognize United Arab Emirates president Sheik Khalifa bin Zayedal-Nahyan, who as emir of Abu Dhabi gave struggling Dubai a $10 billion bailout last month.)So what buildings could be the next to rise up and steal the Burj Khalifa's crown? Here are eight future contenders.1. Burj Mubarak al KabirLocation /// Madinat Al Hareer (City of Silk), KuwaitProjected Height /// 3284 ft(Photograph by Eric Kuhne and Associates)This mammoth structure will rise to exactly 3284 feet, or 1001 meters. The height, in meters, is an allusion to the classic collection of Middle Eastern and South Asian folk tales One Thousand and One Arabian Nights, says London-based architect Eric Kuhne, whose firm designed the tower. To break the kilometer-high mark (which is 3281 feet), the $7 billion-plus Mubarak al Kabir will have three interlocked towers that support the overall structure. These towers, or "blades," pinwheel about a triangular central shaft that holds elevators and mechanical equipment. Each blade twists 45 degrees as it rises, for strength, and expands slightly at the top. This Kuwaiti landmark will therefore place more mass and usable space near its zenith compared to other towers, says Kuhne, to avoid the structure having too thin and flexible a tip. To dissipate high-altitude, tower-buffeting gales that could blow at 150 miles per hour, the Mubarak al Kabir will see the first architectural deployment of vertical ailerons—the normally horizontal flaps airline passengers see on a plane's trailing wing edge that help counter wind disturbances. "They will look like continuous ribbons running vertically along the six leading edges of the three blades," Kuhne says. "As [the ailerons] are constantly moving, and catching the sun while they adjust, sunlight will glint off their surfaces. It will add a gentle rippling reflection to the edges of the blades that will add dynamic sparkle to the tower," Kuhne says. The Burj Mubarak has a projected completion date of 2016.2.1 DubaiProjected Height /// Three towers: 1969 ft, 2625 ft and 3281 ftLocation /// Dubai, United Arab EmiratesBuilding higher also means building wider. Thatis why the 3280-foot 1 Dubai will be built withthree towers. "What tends to happen is as thesebuildings get taller, the base needs to be wider,but it gets to the point that it's just too wide to bea single building and you start to pull things apartor separate them," says Peter Weismantle,director of supertall building technology at AdrianSmith and Gordon Gill Architecture. The smallesttower of 1 Dubai will come in at around 1970 feetand the tallest at about 3280. All three emergefrom a tripedal base architects call the saddle. Acanal will flow between 1 Dubai's three legs,letting boats sail underneath. Further support forthe towers comes from the connectingskybridges where tower residents will be able tocongregate. Designers envision building theskybridges at the saddle and then using a jackingmechanism to hoist them into place. Clearing thesite for the project began in 2008 but has sincebeen put on hold thanks to the state of the world economy. If and when construction begins in earnest, 1 Dubai will take somewhere between seven and 10 years to complete.3.MiapolisProjected Height /// 3000 to 3281ftThe 160-story Miapolis will risenearly 3300 feet on WatsonIsland in Biscayne Bay, just westof Miami Beach and east ofdowntown Miami. The $22 billionMiapolis complex will host anindoor amusement park, luxurycondos and apartments, officespace, a performing arts center,and a marina. With Miapolis,planners hope to demonstrate the potential economic benefits of high-profile real estate: developers say it could bring in nearly a billion in annual tax revenue and pump over twice that into the local economy as visitors flock to South Florida's newest attraction. For now, the project remains on the drawing board at architectural firm Kobi Karp, and there is no shortage of artist'simpressions of the many facets of Miapolis. The designers want the complex to beenvironmentally responsible and intend to have the building receive a LEED Platinum rating by the U.S. Green Buildings Council. Further information about Miapolis is scant for now as developers are tight-lipped about the project, though lead developer Guillermo Socarras says he will be announcing new details in a few weeks. Meanwhile, Socarras is in talks with the Federal Aviation Administration about getting clearance on Miapolis' soaring height, given the proposed site's proximity to Miami International Airport.4.Nakheel TowerLocation /// Dubai, United Arab EmiratesProjected Height /// 3281 to 4593 ftThis cylindrical megatower has eight spires that come to a point at the building's peak. Though an official target height has not been revealed, the Nakheel Tower is likely to crest 3280 feet. Its designers, the international firm Woods Bagot, aim for the Nakheel Tower to be the first true realization of a vertical city. Over 15,000 people will live, work and socialize in this spire with a ground footprint the size of a New York City square block. The placement of support columns is based on a radially symmetrical 16-point star pattern and is inspired by Arabic patternmaking. The pattern makes engineering sense because a symmetrical building bears the load evenly among its structural units, according to a 2009 case study on the Nakheel Tower published in the journal of the Council on Tall Buildings and Urban Habitat. The trickiest part about designing the Nakheel Tower, according to the study, was dealing with so-called vortex shedding from winds, which can cause damaging vibrations. Instead of funneling wind around its metal and glass skin, the Nakheel Tower takes the uncommon approach of having large gaps in the midst of the building, with a double set of slots that let gales pass right through. Every 25 floors or so, big disk-like skybridges bind the towers together and serve as village squares for high-rise dwellers, as in 1 Dubai. Also as in 1 Dubai, the Nakheel Tower's completion date has been held up because of unfavorable market conditions, though some early construction work did get underway before the stall. A completion date has not been announced and the project may never resume.5.Sky City 1000Location /// Tokyo, JapanProjected Height /// 3281 ftThe Takenake Corporationproposed Sky City 1000 backin 1989 to tackle Tokyopopulation-density problems.Tokyo-like congestion promptsa demand for green space andoffice space that vastlyexceeds supply, and alsointroduces a host ofenvironmental and socialissues, from pollution touncomfortably packed commuter trains. Takenake's solution: Build up—way up—and place green spaces in the sky. "The feature of our proposal was making artificial land in the air," says Masato Ujigawa, manager of the engineering department at Takenaka. To achieve this, Takenake will first start with a base that is 1300 feet per side, a footprint that equates to several city blocks (Burj Khalifa's triangular footprint is just 300 feet or so). Then, in accordance with its name, Sky City 1000 will rise a full thousand meters (3281 feet), consisting of 14 levels stacked on top of one another. Each level will act as its own "town," with a park-like plaza area in its center ringed by residences, schools and businesses. The structure would hold 10,000 homes and be used in some capacity by 130,000 people. Construction has not begun on Sky City 1000 since Japan's population has begun shrinking as of 2005, Ujigawa says. Nevertheless, Ujigawa says that ideas originally espoused by the Sky City 1000 project have since been used in more conventional construction. These include concrete reinforced with carbon fibers instead of iron to cut down on weight, andself-contained water-service systems in buildings that treat sewage and reclaim water.6.Bionic TowerLocation /// (Originally Proposed For) Shanghai, ChinaProjected Height /// 4029 ftThe roughly $15 billion Bionic Tower willbreak from traditional engineeringprinciples, introducing radical designelements for the 4029-foot-tall tower,according to Eloy Celaya, an architectwith ECE Arquitecturas and one of threeprincipal Spanish designers of the BionicTower. Instead of vertical foundations,Celaya envisions a "floating foundation"similar to a tree's roots, with a tangle ofmany hundreds of anchors in the ground.For supportive, criscrossing trusses, the Bionic Tower will draw inspiration from bird bones, which are light and hollow. The twelve stacked neighborhoods within this vertical megalopolis will receive water, energy and other supplies by means of 92 vertical columns (much like the xylem and phloem transport systems in vascular plants), which will double as structural supports. Though the concept for the Bionic Tower was originally pitched to Shanghai, China about a decade ago, at present the prospects for this tower being erected someday are iffy.7.Kingdom TowerLocation /// Jeddah, Saudi ArabiaProjected Height /// 3281-plus ftThis skyscraper was initially billed as the Mile-High Tower in 2008, though therecord-setting height ambitions have since been cut by nearly 2000 feet. Updated design plans have not yet been revealed for the Kingdom Tower, but the winner of a design contest between Skidmore, Owings & Merrill and Adrian Smith and Gordon Gill Architecture should be announced in a few weeks. Marshall Gerometta, of the Council on Tall Buildings and Urban Habitat, the group that certifies supertall building heights, says that the Kingdom Tower probably is the best bet in the near term to overtake the Burj Khalifa. Funding appears secured for this building, which will be the centerpiece of a new $27 billion planned urban area in Jeddah, Saudi Arabia, overseen and financed by the Kingdom Holding Company. The first mile-high setup called for the creation of two stabilizing mini-towers to support the main tower. The mini-towers, at nearly 1000 feet each, about the height of the Eiffel Tower, will be dwarfed by the central spire. Many supertall building tops have an "expected" lateral movement of 10 feet or so, and to mitigate this swaying effect, a massive, computer-controlled object called a damper will be placed within the mile-high structure. What the eventual building will look like and how it will be engineered remain open questions, though Gerometta says he heard the Kingdom Tower was going to represent "a new generation of skyscrapers."lennium Challenge TowerLocation /// TBDProjected Height /// 6076 ftThis concept tower has also been referred to as the AlJaber Tower in accordance with its possible placement inKuwait. This tower would soar to a full nautical mile, 1852meters, or over 6000 feet. Italian architect OmeroMarchetti, the founder of the Millennium Challenge 1852project, says "to reach [a marine mile] you cannot useconcrete, orthogonal grids, traditional systems, mortars,[and] cranes." The building would dispense with rightangles and perpendicular planes as these structuralengineering norms make large quantities of cast iron andconcrete "follow an unnatural and twisted geometry,"Marchetti says. He has instead looked to the hexagonalmatrices of snowflakes, which as structurally supportedobjects combine high volume with low weight. Marchettisays that currently three groups of investors in differentparts of the world are interested in making the MillenniumChallenge Tower a reality, a step he believes isnecessary to make a sustainable planet. "I think we havenot a second chance, or if you prefer, we have not asecond planet," Marchetti says. "I tell you that this is thefuture, which is up to us to capture now."八座将要挑战迪拜塔的摩天大楼本月，刚刚登上世界最高建筑新科状元宝座的迪拜哈利法塔盛大揭幕但建筑师们不会就此止步。

GE HealthcareInstructions 71-5016-96 AK HiTrap affinity columns GSTrap FF,1 ml and 5 mlGSTrap™ FF columns are prepacked 1 ml and 5 ml HiTrap™ columns for convenient, one-step purification of glutathione S-transferase (GST) tagged proteins produced using the pGEX series of expression vectors, other glutathione S-transferases and glutathione binding proteins.GST-tagged proteins can be purified directly from pretreated bacterial lysates using GSTrap FF. Tagged proteins are eluted under mild, nondenaturing conditions that preserve protein antigenicity and function.The medium, Glutathione Sepharose™ 4 Fast Flow, is also available as lab packages and is an excellent choice for scale-up.The columns can be operated with a syringe, peristaltic pump or liquid chromatography system such as ÄKTAdesign™ or FPLC™ System.Code No. Product No. supplied 17-5130-01 GSTrap FF 5 × 1 ml17-5130-02 GSTrap FF 2 × 1 ml17-5130-05 GSTrap FF 100 × 1 ml* 17-5131-01 GSTrap FF 1 × 5 ml17-5131-02 GSTrap FF 5 × 5 ml17-5131-05 GSTrap FF 100 × 5 ml* * Special package delivered on specific customer order.ConnectorkitConnectors supplied Usage No. supplied 1/16” male/luer female Connection of syringe to top ofHiTrap column 1 Tubing connector Connection of tubing (e.g. Peristalticflangeless/M6 female Pump P1) to bottom of HiTrap column* 1 Tubing connector Connection of tubing (e.g. Peristalticflangeless/M6 male Pump P1) to top of HiTrap column** 1 Union 1/16” female/ Connection to original FPLC SystemM6 male through bottom of HiTrap column 1 Union M6 female/ Connection to original FPLC System1/16” male through top of HiTrap column 1 Stop plug female, 1/16” Sealing bottom of HiTrap column 2, 5 or 7 * Union 1/16” female/M6 male is also needed.** Union M6 female/1/16” male is also needed.Tables of contents1. Description 32. Operation 53. Scaling up 74. Storage 75. Cleavage of GST-tagged proteins 76. Troubleshooting guide 127. References 178. Ordering information 18p.p.1. DescriptionMedium propertiesGlutathione Sepharose 4 Fast Flow is designed for purification ofglutathione S-transferase (GST) tagged proteins produced using the pGEX series of expression vectors (1), other glutathione S-transferases andglutathione binding proteins. GST-tagged proteins can be purified directly from pretreated bacterial lysates with a one-step method using GSTrap FF. The tagged proteins are eluted under mild, non-denaturing conditions that preserve protein antigenicity and function. The glutathione ligand is coupled via a 10-carbon linker to highly cross-linked 4% agarose. The coupling is optimized to give high binding capacity for GST-tagged proteins and other glutathione binding proteins.The total binding capacity is approximately 10 mg recombinant GST/mlmedium. The dynamic binding capacity will vary depending on the flow rate and the sample. If removal of the GST-tag (a naturally occurring M r 26 000 protein) is required, the tagged protein can be digested with the appropriate site-specific protease while bound to GSTrap FF or, alternatively, after elution. Cleavage on GSTrap FF eliminates the extra step of separating the released protein from GST, since the GST-tag remains bound. The target protein is eluted using binding buffer.ColumnThe columns are made of polypropylene, which is biocompatible and non-interactive with biomolecules. The columns have porous top and bottom frits that allow high flow rates. The columns are delivered with a stopper on the inlet and a snapoff end on the outlet. The separation can be easily achieved using a syringe together with the supplied adaptor, a pump, or a chromatography system such as ÄKTA ™ or FPLC.Note: To prevent leakage it is essential to ensure that the adaptor is tight.Several columns can be connected in series to increase binding capacity. (Backpressure will increase).The column cannot be opened or refilled.The characteristics of GSTrap FF are summarized below.Table . GSTrap FF characteristicsColumn dimensions (i.d. × h) 0.7 × 2.5 cm (1 ml) and 1.6 × 2.5 cm (5 ml)Column volumes 1 ml and 5 ml respectivelyLigand Glutathione and 10-carbon linker armLigand concentration 120–320 µmol glutathione/ml mediumBinding capacity* ≈ 10 mg recombinant glutathioneS-transferase/ml medium GST, M r 26 000 Dynamic binding capacity* ≈ 11 mg GST-tagged protein/ml mediumM r 43 000 (GSTrap FF 1 ml at 1 ml/min) Average particle size 90 µmBead structure Highly cross-linked 4% agaroseMaximum back pressure 0.3 MPa, 3 barMaximum flow rate 4 ml/min and 15 ml/min for 1 and 5 mlcolumns respectivelyRecommended flow rates* Sample loading: 0.2–1 ml/min (1 ml ) and1–5 ml (5 ml)Washing and elution: 1–2 ml/min (1 ml) and 5–10 ml/min (5 ml)Chemical stability All commonly used aqueous buffers, e.g.1 M acetate pH 4.0 and 6 M guanidinehydrochloride for 1 hour at room temperature pH stability pH 3–12Storage temperature + 4 to + 30 °CStorage 20 % ethanol*Note:Binding of GST to glutathione is flow dependent and lower flow rates often increase the binding capacity. This is important during sample loading and elution. Proteincharacteristics, pH and temperature may also affect the binding capacity.p.p. 52. OperationThe columns can be operated with a syringe, peristaltic pump or a liquid chromatography system.Buffer preparationWater and chemicals used for buffer preparation should be of high purity. We recommend filtering the buffers by passing them through a 0.45 µm filter before use.Binding buffer: PBS, pH 7.3 (140 mM NaCl, 2.7 mM KCl, 10 mMNa 2HPO 4, 1.8 mM KH 2PO 4, pH 7.3)Elution Buffer:50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0Note: 1–10 mM DTT can be included in the binding and elution buffers.Sample preparationThe sample should be centrifuged and/or filtered through a 45 µm filter immediately before it is applied to the column. If the sample is too viscous, dilute it with binding buffer to prevent clogging the column.Purification1. Fill the pump tubing or syringe with binding buffer. Connect the columnto the syringe (use the adaptor supplied) or pump tubing “drop to drop” to avoid introducing air into the column.2. Remove the snap-off end at the column outlet.3. Equilibrate the column with 5 column volumes of binding buffer.4. Apply the sample using a syringe fitted to the luer adaptor or bypumping it onto the column. For best results, use a flow rate of0.2–1 ml/min (1 ml column) and 1–5 ml/min (5 ml column) during sample application.p. 65. Wash with 5–10 column volumes of binding buffer or until no materialappears in the effluent. A flow rate of 1–2 ml/min (1 ml column) and 5–10 ml/min (5 ml column) is recommended for washing.6. Elute with 5–10 column volumes of elution buffer. A flow rate of1–2 ml/min (1 ml column) and 5–10 ml/min (5 ml column) is recommended for elution.Note: • One of the most important parameters affecting the bindingof GST-tagged proteins or other glutathione binding proteins to GSTrap FF is the flow rate. Due to the relatively slow binding kinetics between GST and glutathione, it is important to keep the flow rate low during sample application for maximum binding capacity. Protein characteristics, pH and temperature are other factors that may affect the binding capacity.• Volumes and times used for elution may vary among fusionproteins. Additional elutions with higher concentrations of glutathione may be required. Flowthrough, wash and eluted material from the column should be monitored for GST-tagged proteins using SDS-PAGE in combination with Western Blot if necessary.• The GST Detection Module can be used to optimize conditions forelution or to trace steps in the purification of a GST-tagged protein. The Module is designed to identify GST-tagged proteins using either a biochemical or an immunological assay.• The concentration of GST-tagged protein can be estimatedby measuring the absorbance at 280 nm. The GST-tag can be approximated using the conversion; A 280 ≈ 1 corresponds to ~ 0.5 mg/ml.• The concentration of GST-tagged protein may also be determinedby standard chromogenic methods (e.g. Lowry, BCA, and Bradford assays). If Lowry or BCA assays are to be used, the sample must first be buffer exchanged using a HiTrap Desalting 5 ml column, a HiPrep ™ 26/10 Desalting column or dialysed against PBS to remove glutathione, which can interfere with the protein measurement. The Bradford method can be used in the presence of glutathione.• The reuse of GSTrap FF depends on the nature of the sample andshould only be performed with identical samples to prevent cross-contamination.Cleaning GSTrap FFIf the medium appears to be losing binding capacity, it may be due to an accumulation of precipitate, denatured or nonspecifically bound proteins. Removal of precipitated or denatured substances:• Wash with 2 column volumes of 6 M guanidine hydrochloride, immediately followed by 5 column volumes of PBS.Removal of hydrophobically bound substances:• Wash with 3–4 column volumes of 70% ethanol or 2 column volumes of 1% Triton™ X-100 immediately followed by 5 column volumes of PBS. 3. Scaling upFor quick scale-up of purifications, two or three GSTrap FF can be connected in series (backpressure will increase). Further scaling up is easy using the20 ml prepacked GSTPrep™ FF 16/10 column or bulk media packages.4. StorageStore the column at +4 to 30 °C in 20% ethanol.5. Cleavage of GST-tagged proteinsIf removal of the GST-tag is necessary, tagged proteins containing a PreScission™ Protease recognition site, a thrombin recognition site or afactor Xa recognition site may be cleaved either while bound to GSTrap FF or in solution after elution. Cleavage after elution is suggested if optimizationof cleavage conditions is necessary. Samples can easily be removed at various time points and analyzed by SDS-PAGE to estimate the yield, purity and extent of digestion. The amount of protease used, the temperatureand the length of incubation required for complete digestion may varyp. 7p.depending on the fusion partner. Optimal conditions for each fusion should be determined in pilot experiments, e.g. incubation time may be reduced by using higher concentrations of proteolytic enzyme.1. PreScission ProteasePreScission Protease, M r 46 000PreScission cleavage buffer: 50 mM Tris-HCl, 150 mM NaCl,1 mM EDTA, 1 mM dithiothreitol (DTT), pH 7.5PreScission Protease cleavage of GST-tagged protein bound to GSTrap FFAssumption: 8 mg GST-tagged protein bound/ml medium 1. Follow steps 1–5 under “Purification”, (see p. 5).2. Wash GSTrap FF with 10 column volumes of PreScission cleavage buffer.3. Prepare the PreScission Protease mix:GSTrap FF 1 ml column (8 mg GST-tagged protein bound): Mix 80 µl (160 units) of PreScission Protease with 920 µl of PreScission cleavage buffer at +4 °C.GSTrap FF 5 ml column (40 mg GST-tagged protein bound): Mix 400 µl (800 units) of PreScission Protease with 4.6 ml of PreScission cleavage buffer at +4 °C.4. Load the PreScission Protease mix onto the column using a syringe andthe adaptor supplied.Seal the column with the top and bottom stop plugs supplied.5. Incubate the column at +4 °C for 4 hours.6. Fill a syringe with 3 ml (1 ml column) or 15 ml (5 ml column) of PreScissioncleavage buffer. Remove the top and bottom stop plugs. Avoidintroducing air into the column. Elute the column and collect the eluate (0.5 ml-1 ml/tube). The eluate will contain the protein of interest, while the GST moiety of the tagged protein and the PreScission Protease will remain bound to GSTrap FF.PreScission Protease cleavage of eluted GST-tagged protein Assumption: 8 mg GST-tagged protein bound/ml medium1. Follow steps 1–6 under “Purification”, (see page 5).2. Remove the reduced glutathione from the eluate using a quick bufferexchange on HiTrap Desalting, a PD-10 column or HiPrep 26/10 Desalting depending on the sample volume, or dialyse against PreScissioncleavage buffer.3. Add 1 µl (2 U) of PreScission Protease for each 100 µg of tagged proteinin the eluate. If the amount of tagged protein in the eluate has not been determined, add 80 µl (160 units) of PreScission Protease (tagged protein eluted from GSTrap FF 1 ml column) or 400 µl (800 units) of PreScissionProtease (tagged protein eluted from GSTrap FF 5 ml column).4. Incubate at +4 °C for 4 hours.5. Once digestion is complete, apply the sample to an equilibrated GSTrapFF column to remove the GST moiety of the tagged protein and thePreScission Protease. The protein of interest will be found in the flow-through, while the GST moiety of the tagged protein and the PreScission Protease will remain bound to GSTrap FF.. Thrombin37 000Thrombin, MrThrombin cleavage buffer: PBS, pH 7.3Preparation of thrombin solution:1. Dissolve 500 U thrombin in cold 500 µl PBS (1 U/µl).2. Swirl gently to dissolve.3. Freeze as 80 µl aliqouts and keep at –80 °C.Thrombin cleavage of GST-tagged protein bound to GSTrap FF Assumption: 8 mg GST-tagged protein bound/ml medium1. Follow steps 1–5 under “Purification”, (see p. 5).2. Prepare the thrombin mix: GSTrap FF 1 ml column (8 mg GST fusionprotein bound): Mix 80 µl thrombin solution (1 U/µl ) with 920 µl PBS.p. 9GSTrap FF 5 ml column (40 mg GST-tagged protein bound): Mix 400 µlthrombin solution with 4.6 ml PBS.3. Load the thrombin solution onto the column using a syringe and theadaptor supplied. Seal the column with the top and bottom plugssupplied.4. Incubate the column at room temperature (+22 to +25 °C) for 2–16 hours.5. Fill a syringe with 3 ml (1 ml column) or 15 ml (5 ml column) PBS. Removethe top and bottom stop plugs from the column. Avoid introducing airinto the column. Elute the column and collect the eluate (0.5 ml-1 ml/tube). The eluate will contain the protein of interest and thrombin, while the GST moiety of the tagged protein will remain bound to GSTrap FF. Note:After cleavage using thrombin the enzyme can be removed from eluted protein using HiTrap Benzamidine FF (high sub), see orderinginformation.Thrombin cleavage of eluted GST-tagged proteinAssumption: 8 mg GST-tagged protein bound/ml medium1. Follow steps 1–6 under “Purification”, (see p. 5).2. Add 10 µl (10 units) of thrombin solution for each mg of tagged proteinin the eluate. If the amount of tagged protein in the eluate has not been determined, add 80 µl (80 U) thrombin solution (tagged protein elutedfrom GSTrap FF 1 ml column) or 400 µl (400 U) thrombin solution (tagged protein eluted from GSTrap FF 5 ml column).3. Incubate at room temperatue (+22 to 25 °C) for 2–16 hours.4. Once digestion is complete, GST can be removed by first removingglutathione using a quick buffer exchange on HiTrap Desalting, a PD-10 column or HiPrep 26/10 Desalting depending on the sample volume, or dialysing against PBS. Then apply the sample to an equilibrated GSTrap FF column. The purified protein of interest and thrombin will be found in the flow-through.Note:After cleavage using thrombin the enzyme can be removed from eluted protein using HiTrap Benzamidine FF (high sub), see orderinginformation.p. 10. Factor Xa48 000Factor Xa, MrNote:Factor Xa consists of two subunits linked by disulfide bridges. As glutathione can disrupt disulfide bridges, it should be removed fromthe sample prior to the cleavage reaction., pH 7.5 Factor Xa cleavage buffer: 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2 Preparation of factor Xa solution:1. Dissolve 400 U factor Xa in 400 µl cold water (1 U/µl).2. Swirl gently to dissolve.3. Freeze as 80 µl aliqouts and keep at –80°C.Factor Xa cleavage of GST-tagged protein bound to GSTrap FF Assumption: 8 mg GST-tagged protein bound/ml medium1. Follow steps 1–5 under “Purification”, (see p. 5).2. Wash GSTrap FF with 10 column volumes of factor Xa cleavage buffer.3. Prepare the factor Xa mix:GSTrap FF 1 ml column (8 mg GST-tagged protein bound): Mix 80 µl factor Xa solution with 920 µl factor Xa cleavage buffer.GSTrap FF 5 ml column (40 mg GST-tagged protein bound): Mix 400 µlfactor Xa solution with 4.6 ml factor Xa cleavage buffer.4. Load the mix onto the column using a syringe and the adaptor supplied.Seal the column with the top and bottom stop plugs supplied.5. Incubate the column at room temperature (+22 to 25 °C) for 2–16 hours.6. Fill a syringe with 3 ml (1 ml column) or 15 ml (5 ml column) factorXa cleavage buffer. Remove the top and bottom stop plugs from thecolumn. Avoid introducing air into the column. Elute the column andcollect the eluate (0.5 ml-1 ml/tube). The eluate will contain the proteinof interest and factor Xa, while the GST moiety of the tagged protein will remain bound to GSTrap FF.p. 11Note:After cleavage using factor Xa the enzyme can be removed from eluted protein using HiTrap Benzamidine FF (high sub), see orderinginformation.Factor Xa cleavage of eluted GST-tagged proteinAssumption: 8 mg GST-tagged protein bound/ml medium1. Follow steps 1–6 under “Purification”, (see p. 5).2. Remove the reduced glutathione from the eluate using a quick bufferexchange on HiTrap Desalting, a PD-10 column or HiPrep 26/10 Desalting depending on sample volume, or dialyse against factor Xa cleavagebuffer.3. Add 10 units of factor Xa solution for each mg tagged protein in theeluate. If the amount of tagged protein in the eluate has not beendetermined, add 80 µl (80 units) of factor Xa solution (eluted taggedprotein from GSTrap FF 1 ml column) or 400 µl (400 units) of factor Xasolution (eluted tagged protein from GSTrap FF 5 ml column).4. Incubate the column at room temperature (+22 to 25 °C) for 2–16 hours.5. Once digestion is complete, apply the sample to an equilibrated GSTrapFF column to remove the GST moiety . The protein of interest will befound in the flow-through together with factor Xa.Note:After cleavage using factor Xa the enzyme can be removed from eluted protein using HiTrap Benzamidine FF (high sub), see orderinginformation.6. Troubleshooting guideConsult the GST Gene Fusion System Handbook (1) for more detailedinformation and pGEX instructions regarding troubleshootingrecommendations for expression, fermentation and solubilization.GST-tagged protein does not bind to GSTrap FF• GST-tagged protein denatured by sonication: Too extensive sonication can denature the tagged protein and prevent it binding to GSTrap FF. Use mild sonication conditions during cell lysis.p. 1• Add DTT prior to cell lysis: Adding DTT to a final concentration of 1–10 mM prior to cell lysis may significantly increase binding of someGST-tagged proteins to GSTrap FF.•Test the binding of GST from parental pGEX: Prepare a sonicate of cells harboring the parental pGEX plasmid and check binding to the matrix.If GST produced from the parental plasmid binds with high affinity,the fusion partner may have altered the conformation of GST, therebyreducing its affinity. Adequate results may be obtained by reducing the temperature used for binding to +4°C, and by limiting column washing.•Equilibrate GSTrap FF before use: Binding of GST-tagged proteins to GSTrap FF is not efficient at pH less than 6.5 or greater than 8. Check that the GSTrap FF column has been equilibrated with a buffer pH 6.5 to 8.0(e.g. PBS) before the cell lysate is applied.• Use a fresh GSTrap FF: If the GSTrap FF column has already been used several times, it may be necessary to use a new GSTrap FF column. Seealso “Cleaning GSTrap FF”.• Decrease flow rate during sample load, see note p. 6.GST-tagged protein is not eluted efficiently from GSTrap FF• Increase the time used for elution: Decrease the flow during elution.•Increase the volume of elution buffer: Sometimes, especially after on-column cleavage of fusion protein, a larger volume of buffer may be necessary to elute the fusion protein.• Increase the concentration of glutathione in the elution buffer: The10 mM recommended in this protocol should be sufficient for mostapplications, but exceptions exist. Try 50 mM Tris- HCl, 20–40 mMreduced glutathione, pH 8.0 as elution buffer.•Increase the pH of the elution buffer: A low pH may limit elution from GSTrap FF. Increasing the pH of the elution buffer to pH 8–9 may improve elution without requiring an increase in the concentration of glutathione used for elution.• Increase the ionic strength of the elution buffer: Adding 0.1–0.2 M NaCl to the elution buffer may also improve results.p. 1p. 1• Add a non-ionic detergent to the elution buffer: Non-specifichydrophobic interactions may prevent solubilization and elution of fusion proteins from GSTrap FF. Adding a non-ionic detergent may improve results. Adding 0.1% Triton X-100 or 2% N-octylglucosid can significantly improve elution of some GST-tagged proteins.Multiple bands are observed after electrophoresis/Western Blotting analysis of eluted target protein• M r 70 000 protein co-purifies with the GST-tagged protein:The M r 70 000 protein is probably a protein product of the E. coli genednaK. This protein is involved in protein folding in E. coli . It has beenreported that this association can be disrupted by incubating the fusion protein in 50 mM Tris-HCl, 2 mM ATP, 10 mM MgSO 4, pH 7.4 for 10 min. at +37 °C prior to loading on GSTrap FF.Alternatively, remove the DnaK protein by passing the tagged protein solution through ATP-agarose or by ion exchange.• Add a protease inhibitor: Multiple bands may be a result of partialdegradation of tagged proteins by proteases. Adding 1 mM PMSF to the lysis solution may improve results. A nontoxic, water-soluble alternative to PMSF is 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF), commercially available as Pefabloc ™ SC from Boehringer Mannheim.Note: Serine protease inhibitors must be removed prior to cleavage bythrombin or factor Xa. PreScission Protease is not a consensus serine protease and is insensitive to many of the protease inhibitors tested at GE Healthcare.• Use a protease-deficient host: Multiple bands may be the result ofproteolysis in the host bacteria. If this is the case, the use of a host-deficient strain may be required (e.g. lon - or ompT ). E. coli BL21 is provided with the pGEX vectors. This strain is ompT.• Decrease sonication: Cell disruption is apparent by partial clearing ofthe suspension and can be checked by microscopic examination. Adding lysozyme (0.1 volume of a 10 mg/ml lysozyme solution in 25 mM Tris-HCl, pH 8.0) prior to sonication may improve results. Avoid frothing as thisp. 15may denature the fusion protein. Over-sonication can also lead to the co-purification of host proteins with the GST-tagged protein.• Include an additional purification step: Additional bands maybe caused by the co-purification of a variety of proteins known as chaperonins, which are involved in the correct folding of nascent proteins in E. coli . These include, but may not be limited to: DnaK (M r ~ 70 000), DnaJ (Mr ~ 37 000), GrpE (M r ~ 40 000), GroEL (M r ~ 57 000) and GroES (M r ~ 10 000). Several methods for purifying GST-tagged proteins from these co-purifying proteins have been described.• Cross-adsorb antibody with E. coli proteins: Depending on the sourceof the anti-GST antibody, it may contain antibodies that react with various E. coli proteins that may be present in your fusion protein sample. Cross-adsorb the antibody with an E. coli sonicate to remove anti-E. coli antibodies from the preparation. Anti-GST antibody from GE Healthcare has been cross-adsorbed against E. coli proteins and tested for its lack of non-specific background binding in Western Blots.Incomplete cleavage of GST-tagged proteins• The PreScission Protease, thrombin or factor Xa to tagged proteinratios are incorrect: Check the amount of tagged protein in thedigestion. Note that the capacity of GSTrap FF for GST is approximately 10 mg/ml medium. In most purifications, however, the matrix is not saturated with tagged protein.Ratios: PreScission protease, at least 10 units/mg tagged protein.Thrombin, at least 10 units/mg tagged protein. One cleavage unit of thrombin from GE Healthcare digests ≥ 90% of 100 µg of a test tagged protein in 16 hours at +22 °C.Factor Xa, at least 1% (w/w) tagged protein. For some tagged proteins, up to 5% factor Xa can be used. The optimum amount must be determined empirically.In some cases, a tagged protein concentration of 1 mg/ ml has been found to give optimal results. Adding ≤ 0.5% (w/v) to the reaction buffer can significantly improve factor Xa cleavage with some tagged proteins. Various concentrations of SDS should be tested to find the optimum concentration.p. 16• Increase incubation time and enzyme concentration: For PreScissionProtease, thrombin or factor Xa, increase the reaction time to 20 hours or more if the tagged protein is not degraded by extensive incubation. The amount of enzymes can also be increased.• Verify the presence of specific cleavage sites: Check the DNA sequenceof the construct. Compare it with a known sequence and verify that the different specific cleavage sites for the enzyme used have not been altered during the cloning of your tagged protein.Ensure that cleavage enzyme inhibitors are absent• PreScission Protease: Buffer exchange or dialyse the tagged proteinagainst 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.5 before cleavage.• Factor Xa: Buffer exchange on HiTrap Desalting, a PD-10 column orHiPrep 26/10 Desalting depending on the sample volume, or dialyse against 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl 2, pH 7.5.• Factor Xa is not properly activated: Functional factor Xa requiresactivation of factor X with Russell’s viper venom. Activation conditions are a ratio of Russell’s viper venom to factor Xa of 1% in 8 mM Tris-HCl, 70 mM NaCl, 8 mM CaCl 2, pH 8.0. Incubate at +37 °C for 5 min. Factor Xa from GE Healthcare has been preactivated by this procedure.• The first amino acid after the factor Xa recognition sequence is Argor Pro: Check the sequence of the fusion partner to be sure that the first three nucleotides after the factor Xa recognition sequence do not code for Arg or Pro.Multiple bands are observed on SDS gels following enzyme cleavage:• Determine when the bands appear: Test to be certain that additionalbands are not present prior to PreScission Protease, thrombin or factor Xa cleavage. Such bands may be the result of proteolysis in the host bacteria.• Tagged partner may contain recognition sequences for PreScissionProtease, thrombin or factor Xa: Check the sequences. See the GST Gene Fusion System Handbook (1) for details.7. References1. GST Gene Fusion System Handbook, GE Healthcare, Code No. 18-1157-582. Rapid purification of GST-fusion proteins from large sample volumes.Miniposter, 18-1139-51, GE Healthcare.3. Efficient, rapid protein purification and on-column cleavage usingGSTrap FF columns. Application Note, 18-1146-70, GE Healthcare.4. Purification of GST-fusion proteins, on-column cleavage and sampleclean-up. Miniposter, 18-1150-20, GE Healthcare.5. Dian, C., et al, J of chromatography B, 769 (1), 133–144 (2002)6. Strategies for the purification and on-column cleavage of glutathioneS-transferase fusion target proteins. J of Chromatography B, 2002. 769(1): 133–144. Dian, C., et al.For more information about HiTrap columns and updated reference list forthe use of GSTrap FF columns, (Code No. 18-1156-67) visit/hitrapp. 17。

ConnectionsTeaching ToolkitA Teaching Guide for Structural Steel ConnectionsPerry S.Green,Ph.D.Thomas Sputo,Ph.D.,P.E.Patrick VeltriThis connection design tool kit for students is based on the original steel sculpture designed by Duane S. Ellifritt, P.E., Ph.D., Professor Emeritus of Civil Engineering at the Uni-versity of Florida. The tool kit includes this teaching guide, a 3D CAD file of the steel sculpture, and a shear connection calculator tool. The teaching guide contains drawings and photographs of each connection depicted on the steel sculp-ture, the CAD file is a 3D AutoCAD® model of the steel sculpture with complete dimensions and details, and the cal-culator tool is a series of MathCAD® worksheets that enables the user to perform a comprehensive check of all required limit states.The tool kit is intended as a supplement to, not a replace-ment for, the information and data presented in the Ameri-can Institute of Steel Construction’s Manual of Steel Construction, Load & Resistance Factor Design, Third Edi-tion, hereafter, referred to as the AISC Manual. The goal of the tool kit is to assist students and educators in both learn-ing and teaching basic structural steel connection design by visualization tools and software application.All information and data presented in any and all parts of the teaching tool kit are for educational purposes only. Although the steel sculpture depicts numerous connections, it is by no means all-inclusive. There are many ways to connect structural steel members together.In teaching engineering students in an introductory course in steel design, often the topic of connections is put off until the end of the course if covered at all. Then with the crush of all the other pressures leading up to the end of the semes-ter, even these few weeks get squeezed until connections are lucky to be addressed for two or three lectures. One reason for slighting connections in beginning steel design, other than time constraints, is that they are sometimes viewed as a “detailing problem” best left to the fabricator. Or, the mis-taken view is taken that connections get standardized, espe-cially shear connections, so there is little creativity needed in their design and engineers view it as a poor use of their time. The AISC Manual has tables and detailing informa-tion on many standard types of connections, so the process is simplified to selecting a tabulated connection that will carry the design load. Many times, the engineer will simply indicate the load to be transmitted on the design drawings and the fabricator will select an appropriate connection. Yet connections are the glue that holds the structure together and, standardized and routine as many of them may seem, it is very important for a structural engineer to under-stand their behavior and design. Historically, most major structural failures have been due to some kind of connection failure. Connections are always designed as planar, two-dimensional elements, even though they have definite three-dimensional behavior. Students who have never been around construction sites to see steel being erected have a difficult time visualizing this three-dimensional character. Try explaining to a student the behavior of a shop-welded, field-bolted double-angle shear connection, where the out-standing legs are made purposely to flex under load and approximate a true pinned connection. Textbooks generally show orthogonal views of such connections, but still many students have trouble in “seeing” the real connection.In the summer of 1985, after seeing the inability of many students to visualize even simple connections, Dr. Ellifritt began to search for a way to make connections more real for them. Field trips were one alternative, but the availability of these is intermittent and with all the problems of liability, some construction managers are not too anxious to have a group of students around the jobsite. Thought was given to building some scale models of connections and bringing them into the classroom, but these would be heavy to move around and one would have the additional problem storing them all when they were not in use.The eventual solution was to create a steel sculpture that would be an attractive addition to the public art already on campus, something that would symbolize engineering in general, and that could also function as a teaching aid. It was completed and erected in October 1986, and is used every semester to show students real connections and real steel members in full scale.Since that time, many other universities have requested a copy of the plans from the University of Florida and have built similar structures on their campuses.PREFACEConnections Teaching Toolkit • iConnection design in an introductory steel course is often difficult to effectively communicate. Time constraints and priority of certain other topics over connection design also tend to inhibit sufficient treatment of connection design. The Steel Connections Teaching tool kit is an attempt to effectively incorporate the fundamentals of steel connection design into a first course in steel design. The tool kit addresses three broad issues that arise when teaching stu-dents steel connection design: visualization, load paths, and limit states.In structural analysis classes, students are shown ideal-ized structures. Simple lines represent beams and columns, while pins, hinges, and fixed supports characterize connec-tions. However, real structures are composed of beams, girders, and columns, all joined together through bolting or welding of plates and angles. It is no wonder that students have trouble visualizing and understanding the true three-dimensional nature of connections!The steel sculpture provides a convenient means by which full-scale steel connections may be shown to stu-dents. The steel sculpture exhibits over 20 different connec-tions commonly used in steel construction today. It is an exceptional teaching instrument to illustrate structural steel connections. The steel sculpture’s merit is nationally recog-nized as more than 90 university campuses now have a steel sculpture modeled after Dr. Ellifritt’s original design.In addition to the steel sculpture, this booklet provides illustrations, and each connection has a short description associated with it.The steel sculpture and the booklet “show” steel connec-tions, but both are qualitative in nature. The steel sculpture’s connections are simply illustrative examples. The connec-tions on the steel sculpture were not designed to satisfy any particular strength or serviceability limit state of the AISC Specification. Also, the narratives in the guide give only cursory descriptions, with limited practical engineering information.The main goals of this Guide are to address the issues of visualization, load paths, and limit states associated with steel connections. The guide is intended to be a teaching tool and supplement the AISC Manual of Steel Construction LRFD 3rd Edition. It is intended to demonstrate to the stu-dent the intricacies of analysis and design for steel connec-tions.Chapters in this guide are arranged based on the types of connections. Each connection is described discussing vari-ous issues and concerns regarding the design, erectability, and performance of the specific connection. Furthermore,every connection that is illustrated by the steel sculpture has multiple photos and a data figure. The data figure has tables of information and CAD-based illustrations and views. Each figure has two tables, the first table lists the applicable limit states for the particular connection, and the second table provides a list of notes that are informative statements or address issues about the connection. The views typically include a large isometric view that highlights the particular location of the connection relative to the steel sculpture as well as a few orthogonal elevations of the connection itself. In addition to the simple views of the connections provided in the figures, also included are fully detailed and dimen-sioned drawings. These views were produced from the full 3D CAD model developed from the original, manually drafted shop drawings of the steel sculpture.The guide covers the most common types of steel con-nections used in practice, however more emphasis has been placed on shear connections. There are more shear connec-tions on the steel sculpture than all other types combined. In addition to the shear connection descriptions, drawings, and photos, MathCAD® worksheets are used to present some design and analysis examples of the shear connections found on the steel sculpture.The illustrations, photos, and particularly the detail draw-ings that are in the teaching guide tend to aid visualization by students. However, the 3D CAD model is the primary means by which the student can learn to properly visualize connections. The 3D model has been developed in the com-monly used AutoCAD “dwg” format. The model can be loaded in AutoCAD or any Autodesk or other compatible 3D visualization application. The student can rotate, pan and zoom to a view of preference.The issue of limit states and load paths as they apply to steel connections is addressed by the illustrations and narra-tive text in the guide. To facilitate a more inclusive under-standing of shear connections, a series of MathCAD®worksheets has been developed to perform complete analy-sis for six different types of shear connections. As an analy-sis application, the worksheets require load and the connection properties as input. Returned as output are two tables. The first table lists potential limit states and returns either the strength of the connection based on a particular limit state or “NA” denoting the limit state is not applicable to that connection type. The second table lists connection specific and general design checks and returns the condition “OK” meaning a satisfactory value, “NA” meaning the check is not applicable to that connection type, or a phrase describing the reason for an unsatisfactory check (e.g.INTRODUCTIONii• Connections Teaching Toolkit“Beam web encroaches fillet of tee”). The student is encouraged to explore the programming inside these work-sheets. Without such exploration, the worksheets represent “black boxes.” The programming must be explored and understood for the benefits of these worksheets to be real-ized.A complete user’s guide for these worksheets can be found in Appendix A. Contained in the guide is one exam-ple for each type of shear connection illustrated by the steel sculpture. Each example presents a simple design problem and provides a demonstration of the use of the worksheet. AppendixB provides a list of references that includes manuals and specifications, textbooks, and AISC engineer-ing journal papers for students interested in further informa-tion regarding structural steel connections.Many Thanks to the following people who aided in the development of this teaching aid and the steel sculpture Steel Teaching Steel Sculpture CreatorDuane Ellifritt, Ph.D., P.E.Original Fabrication DrawingsKun-Young Chiu, Kun-Young Chiu & AssociatesSteel Sculpture Fabrication and ErectionSteel Fabricators, Inc.Steel Sculpture Funding Steel Fabricators, Inc.Teaching tool kit Production StaffPerry S. Green, Ph.D.Thomas Sputo, Ph.D., P.E.Patrick VeltriShear Connection MathCAD® WorksheetsPatrick VeltriAutoCAD Drawings & 3D ModelPatrick VeltriPhotographsPatrick VeltriPerry S. Green, Ph.D.Proofreading and TypesettingAshley ByrneTeaching tool kit FundingAmerican Institute of Steel ConstructionConnections Teaching Toolkit • iiiPreface...............................................................................i. Introduction.....................................................................ii. Chapter 1.The Steel SculptureDesign DrawingsGeneral Notes........................................................1-2 North Elevation.....................................................1-3 South Elevation.....................................................1-4 East Elevation........................................................1-5 West Elevation.......................................................1-6 Chapter 2. Limit StatesBlock Shear Rupture.............................................2-1 Bolt Bearing..........................................................2-2 Bolt Shear..............................................................2-2 Bolt Tension Fracture............................................2-2 Concentrated Forces..............................................2-3 Flexural Yielding...................................................2-4 Prying Action.........................................................2-4 Shear Yielding and Shear Rupture........................2-4 Tension Yielding and Tension Rupture.................2-5 Weld Shear............................................................2-6 Whitmore Section Yielding / Buckling.................2-6 Chapter 3. Joining Steel MembersStructural Bolting..................................................3-1 Welding..................................................................3-2 Chapter 4. Simple Shear ConnectionsShear Connection Examplesand MathCAD worksheets....................................4-1 Double-Angle Connection.....................................4-3 Shear End-Plate Connection...............................4-12 Unstiffened Seated Connection...........................4-12 Single-Plate (Shear Tab) Connection..................4-18 Single-Angle Connection....................................4-18 Tee Shear Connection..........................................4-20Chapter 5: Moment ConnectionsFlange Plated Connections....................................5-1 Directly Welded Flange Connections....................5-5 Extended End Plate Connections..........................5-5 Moment Splice Connections.................................5-7 Chapter 6: Column ConnectionsColumn Splice.......................................................6-1 Base Plates.............................................................6-3 Chapter 7: Miscellaneous ConnectionsClevises.................................................................7-1 Skewed Connection (Bent Plate)..........................7-3 Open Web Steel Joist.............................................7-6 Cold Formed Roof Purlin......................................7-6 Shear Stud Connectors..........................................7-6 Truss Connections.................................................7-6 Chapter 8. Closing RemarksAppendix A. MathCAD WorksheetsUser’s GuideAppendix B. Sources for Additional SteelConnection InformationTABLE OF CONTENTSiv• Connections Teaching ToolkitAs a structure, the steel sculpture consists of 25 steel mem-bers, 43 connection elements, over 26 weld groups, and more than 144 individual bolts. As a piece of art, the steel sculpture is an innovative aesthetic composition of multi-form steel members, united by an assortment of steel ele-ments demonstrating popular attachment methods.At first glance, the arrangement of members and connec-tions on the steel sculpture may seem complex and unorgan-ized. However, upon closer inspection it becomes apparent that the position of the members and connections were methodically designed to illustrate several specific framing and connection issues. The drawings, photos, and illustra-tions best describe the position of the members and connec-tions on the steel sculpture on subsequent pages. The drawings are based on a 3D model of the sculpture. There are four complete elevations of the sculpture followed by thirteen layout drawings showing each connection on the sculpture. Each member and component is fully detailed and dimensioned. A bill of material is included for each lay-out drawing.In general terms, the steel sculpture is a tree-like structure in both the physical and hierarchical sense. A central col-umn, roughly 13 ft tall is comprised of two shafts spliced together 7 ft -6in. from the base. Both shafts are W12-series cross-sections. The upper, lighter section is a W12×106 and the lower, heavier section is a W12×170. Each shaft of the column has four faces (two flanges and two sides of the web) and each face is labeled according to its orientation (North, South, East, or West). A major connection is made to each face of the upper and lower shafts. Seven of the eight faces have a girder-to-column connection while the eighth face supports a truss (partial). Two short beams frame to the web of each girder near their cantilevered end. Thus, the steel sculpture does indeed resemble a tree “branching” out to lighter and shorter members.The upper shaft girder-to-column connections and all of the beam-to-girder connections are simple shear connec-tions. The simply supported girder-to-column connections on the upper shaft are all propped cantilevers of some form. The east-end upper girder, (Girder B8)* is supported by the pipe column that acts as a compression strut, transferring load to the lower girder (Girder B4). A tension rod and cle-vis support the upper west girder (Girder B6). The channel shaped brace (Beam B5A) spans diagonally across two girders (Girder B5 and Girder B8). This channel is sup-ported by the south girder (Girder B5) and also provides support for the east girder (Girder B8).The enclosed CD contains 18 CAD drawings of the steel connections sculpture which may serve as a useful graphi-cal teaching aid.*The identification/labeling scheme for beams, columns, and girders with respect to the drawings included in this document is as follows:CHAPTER 1The Steel Sculpture•Columns have two character labels. The first characteris a “C” and the second character is a number.•Girders have two character labels. The first character isa “B” and the second character is a number.•Beams have three character labels. Like girders, thefirst character is a “B” and the second character is anumber. Since two beams frame into the web of eachgirder, the third character is either an “A” or “B” iden-tifying that the beam frames into either the “A” or “B”side of the girder.•Plates have two character labels that are both arelower-case letters. The first character is a “p”.•Angles have two character labels that are both lower-case letters. The first character is an “a”.Connections Teaching Toolkit • 1-1GENERAL NOTES (U.N.0.)ABBREVIATIONS1-2• Connections Teaching ToolkitFigure 2-1. Block Shear Rupture Limit State (Photo by J.A. Swanson and R. Leon, courtesy ofGeorgia Institute of Technology)Connections Teaching Toolkit • 2-1BSFigure 2-2. Bolt Bearing Limit State(Photo by J.A. Swanson and R. Leon, courtesy of Georgia Institute of Technology)Figure 2-3. Bolt Shear Limit StateFigure 2-4.Bolt Tension Fracture Limit State (Photo by J.A. Swanson and R. Leon, courtesy of Georgia Institute of Technology)2-2• Connections Teaching ToolkitConnections Teaching Toolkit • 2-3Figure 2-5. Flange Local Bending Limit StateFigure 2-6. Web Crippling Limit State Figure 2-7. Web Local Buckling Limit State(SAC Project)2-4• Connections Teaching ToolkitFigure 2-9 Tee Stem Deformation (Astaneh, A., Nader, M.N., 1989)Figure 2-10 Seat Angle Deformation(Yang, W.H. et al., 1997)Figure 2-8 Web Local Yielding Limit State(SAC Project)Figure 2-12.Shear Yielding Limit StateFigure 2-11.Prying Action Limit StateConnections Teaching Toolkit • 2-5Figure 2-14.Tension Fracture Limit StateFigure 2-15.Weld Shear Limit State2-6• Connections Teaching ToolkitFigure 2-16. Whitmore Section Yielding/Buckling Limit StateConnections Teaching Toolkit • 2-7In current construction practice, steel members are joined by either bolting or welding. When fabricating steel for erection, most connections have the connecting material attached to one member in the fabrication shop and the other member(s) attached in the field during erection. This helps simplify shipping and makes erection faster. Welding that may be required on a connection is preferably performed in the more-easily controlled environment of the fabrication shop. If a connection is bolted on one side and welded on the other, the welded side will usually be the shop connec-tion and the bolted connection will be the field connection. The use of either bolting or welding has certain advan-tages and disadvantages. Bolting requires either the punch-ing or drilling of holes in all the plies of material that are to be joined. These holes may be a standard size, oversized, short-slotted, or long-slotted depending on the type of con-nection. It is not unusual to have one ply of material pre-pared with a standard hole while another ply of the connection is prepared with a slotted hole. This practice is common in buildings having all bolted connections since it allows for easier and faster erection of the structural fram-ing.Welding will eliminate the need for punching or drilling the plies of material that will make up the connection, how-ever the labor associated with welding requires a greater level of skill than installing the bolts. Welding requires a highly skilled tradesman who is trained and qualified to make the particular welds called for in a given connection configuration. He or she needs to be trained to make the varying degrees of surface preparation required depending on the type of weld specified, the position that is needed to properly make the weld, the material thickness of the parts to be joined, the preheat temperature of the parts (if neces-sary), and many other variables.STRUCTURAL BOLTINGStructural bolting was the logical engineering evolution from riveting. Riveting became obsolete as the cost of installed high-strength structural bolts became competitive with the cost associated with the four or five skilled trades-men needed for a riveting crew. The Specification for Structural Joints Using ASTM A325 or A490 Bolts, pub-lished by the Research Council on Structural Connections (RCSC, 2000) has been incorporated by reference into the AISC Load and Resistance Factor Design Specification for Structural Steel Buildings. Many of the bolting standards are based on work reported by in the Guide to Design Cri-teria for Bolted and Riveted Joints, (Kulak, Fisher and Struik, 1987).High strength bolts can be either snug tightened or pre-tensioned. When bolts are installed in a snug-tightened con-dition the joint is said to be in bearing as the plies of joined material bear directly on the bolts. This assumes that the shank of the bolt provides load transfer from one ply to the next through direct contact. Bearing connections can be specified with either the threads included (N) or excluded (X) from the shear plane. Allowing threads to be included in the shear planes results in a shear strength about 25% less than if the threads are specified as excluded from the shear plane(s). However, appropriate care must be taken to spec-ify bolt lengths such that the threads are excluded in the as-built condition if the bolts are indeed specified as threads excluded.In pretensioned connections, the bolts act like clamps holding the plies of material together. The clamping force is due to the pretension in the bolts created by properly tightening of the nuts on the bolts. However, the load trans-fer is still in bearing like for snug-tightened joints.The initial load transfer is achieved by friction between the faying or contact surfaces of the plies of material being joined, due to the clamping force of the bolts being normal to the direction of the load. For slip-critical joints, the bolts are pretensioned and the faying surfaces are prepared to achieve a minimum slip resistance. The reliance on friction between the plies for load transfer means that the surface condition of the parts has an impact on the initial strength of slip-critical connections. The strength of slip-critical con-nections is directly proportional to the mean slip coefficient. Coatings such as paint and galvanizing tend to reduce the mean slip coefficient.The two most common grades of bolts available for struc-tural steel connections are designated ASTM A325 and ASTM A490. The use of A307 bolts is no longer that com-mon except for the ½-in. diameter size where they are still sometimes used in connections not requiring a pretensioned installation or for low levels of load. A307 bolts have a 60 ksi minimum tensile strength. A325 and A490 bolts are des-ignated high-strength bolts. A325 bolts have a 120 ksi min-imum tensile strength and are permitted to be galvanized, while A490 bolts have a 150 ksi minimum tensile strength, but are not permitted to be galvanized due to hydrogen embrittlement concerns. High strength bolts are available in sizes from ½- to 1½-in. diameters in 1/8in. increments and can be ordered in lengths from 1½ to 8 inches in ¼ in. incre-ments.CHAPTER 3Joining Steel MembersConnections Teaching Toolkit • 3-1When a pretensioned installation is required, four instal-lation methods are available: turn-of-the-nut, calibrated wrench, twist off bolt, and direct tension indicator methods. The turn-of-the-nut method involves first tightening the nut to the snug tight condition, then subsequently turning the nut a specific amount based on the size and grade of the bolt to develop the required pretension. The calibrated wrench method involves using a torque applied to the bolt to obtain the required level pretension. A torque wrench is calibrated to stall at the required tension for the bolt. Twist-off bolts have a splined end that twists off when the torque corre-sponding to the proper pretension is achieved. ASTM F1852 is the equivalent specification for A325 “twist-off”bolts. Currently, there is no ASTM specification equivalent for A490 tension control bolts. Direct tension indicators (DTIs) are special washers with raised divots on one face. When the bolt is installed, the divots compress to a certain level. The amount of compression must then be checked with a feeler gage.WELDINGWelding is the process of fusing multiple pieces of metal together by heating the metal to a liquid state. Welding can often simplify an otherwise complicated joint, when com-pared to bolting. However, welds are subject to size and length limitations depending on the thickness of the materi-als and the geometry of the pieces being joined. Further-more, welding should be preferably performed on bare metal. Paint and galvanizing should be absent from the area on the metal that is to be welded.Guidelines for welded construction are published by the American Welding Society (AWS) in AWS D1.1 Structural Welding Code-Steel. These provisions have been adopted by the AISC in the Load and Resistance Factor Design Specification for Structural Steel Buildings.Several welding processes are available for joining struc-tural steel. The selection of a process is due largely to suit-ability and economic issues rather than strength. The most common weld processes are Shielded Metal Arc Welding (SMAW), Gas Metal Arc Welding (GMAW), Flux Core Arc Welding (FCAW), and Submerged Arc Welding (SAW). SMAW uses an electrode coated with a material that vaporizes and shields the weld metal to prevent oxidation. The coated electrode is consumable and can be deposited in any position. SMAW is commonly referred to as stick welding.GMAW and FCAW are similar weld processes that use a wire electrode that is fed by a coil to a gun-shaped electrode holder. The main difference between the processes is in the method of weld shielding. GMAW uses an externally sup-plied gas mixture while FCAW has a hollow electrode with flux material in the core that generates a gas shield or a flux shield when the weld is made. GMAW and FCAW can be deposited in all positions and have a relatively fast deposit rate compared to other processes.Figure 3-1. Structural Fastener - Bolt, Nut and Washer Figure 3-2. Direct Tension Indicators and Feeler Gages Figure 3-3. Structural Fastener - Twist-off Bolt3-2• Connections Teaching Toolkit。

镍柱纯化HisTrapHPGE HealthcareInstructions 71-5027-68 AH HisTrap affinity columns HisTrap HP, 1 ml and 5 mlHisTrap? HP is a ready to use column, prepacked with precharged Ni Sepharose? High Performance. This prepacked column is ideal for preparative purification of Histidine-tagged recombinant proteins by immobilized metal ion affinity chromatography (IMAC).The special design of the column, together with the high-performance matrix of the Ni Sepharose medium, provides fast, simple, and easy separations in a convenient format.Ni Sepharose High Performance has low nickel (Ni2+) ion leakage and is compatible with a wide range of additives used in protein purification. HP columns can be operated with a syringe, peristaltic pump, or liquid chromatography system such as ?KTA? design chromatography systems.CAUTION! Contains nickel. May produce an allergic reaction..1Pack size available by special order.Connector kit1Union 1/16” female/M6 male is also needed.2Union M6 female/1/16” male is also needed.Table of contents1.Description..............................................................................32.General considerations.....................................................63.Operation.................................................................................74.Optimization........................................................................115.Stripping and recharging..............................................126.Cleaning-in-place.............................................................137.Scaling-up............................................................................138.Storage..................................................................................149.Troubleshooting................................................................1410.Intended use.......................................................................1711.Ordering Information (18)Code No. Product No. supplied 17-5247-01 HisTrap HP 5 × 1 ml 17-5247-05 HisTrap HP 100 × 1 ml 117-5248-01 HisTrap HP 1 × 5 ml 17-5248-02 HisTrap HP 5 × 5 ml 17-5248-05HisTrap HP 100 × 5 ml 1Connectors supplied Usage No. supplied 1/16” male/luer femaleConnection of syringe to top of HiTrap column1Tubing connector flangeless/M6 femaleConnection of tubing (e.g. Peristaltic Pump P1) to bottom of HiTrap column 11Tubing connector flangeless/M6 male Connection of tubing (e.g. Peristaltic Pump P1) to top of HiTrap column 21Union 1/16” female/M6 male Connection to original FPLC? System through bottom of HiTrap column1Union M6 female/1/16” maleConnection to original FPLC System through top of HiTrap column 1Stop plug female, 1/16”Sealing bottom of HiTrap column 2, 5 or 71DescriptionMedium propertiesHisTrap HP 1 ml and 5 ml columns are prepacked with NiSepharose High Performance, which consists of 34 µm highlycross-linked agarose beads with an immobilized chelating group.The medium has then been charged with Ni2+-ions.Several amino acids, for example histidine, form complexes with many metal ions. Ni Sepharose High Performance selectively binds proteins if suitable complex-forming amino acid residues areexposed on the protein surface.Additional histidines, such as in the case of (histidine)6 -tag,increase affinity for Ni2+ and generally make the histidine-tagged protein the strongest binder among other proteins in for example an E. coli extract.Column propertiesHisTrap HP columns are made of biocompatible polypropylenethat does not interact with biomolecules. Columns are deliveredwith a stopper on the inlet and a snap-off end on the outlet. Thecolumns have porous top and bottom frits that allow high flowrates. They cannot be opened or refilled.Columns can be operated with either a syringe and the supplied luer connector, a peristaltic pump, or a chromatography systemsuch as ?KTA design.Note:To prevent leakage, ensure that the connector is tight.Table 1. HisTrap HP characteristics.1Dynamic binding capacity conditions:Note: Dynamic binding capacity is protein-dependent.2H 2O at room temperature 3Ni 2+-stripped mediumMatrixHighly cross-linked spherical agarose, 6%Average particle size 34 µmMetal ion capacity ~ 15 µmol Ni 2+/ml mediumDynamic binding capacity 1 At least 40 mg (histidine)6-tagged protein/ml medium Column volumes 1 ml or 5 ml Column dimensions i.d. × H:0.7 × 2.5 cm (1 ml) 1.6 × 2.5 cm (5 ml)Recommended flow rate 1 and 5 ml/min for 1 and 5 ml column respectivelyMax. flow rates 4 and 20 ml/min for 1 and 5 ml column respectively Max back pressure 20.3 MPa, 3 barCompatibility during use Stable in all commonly used buffers, reducingagents, denaturants, and detergents (s ee Table 2)Chemical stability 30.01 M HCl, 0.1 M NaOH. Tested for 1 week at 40°C.1 M NaOH, 70% acetic acid. Tested for 12 hours.2% SDS. Tested for 1 hour.30% 2-propanol. Tested for 30 min.Avoid in buffers Chelating agents, e.g. EDTA, EGTA, citrate (see Table 2)pH stability 3short term (at least 2 hours): 2 to 14 long term (≤ 1 week): 3 to 12Storage 20% ethanol Storage temperature4°C to 30°CSample:1 mg/ml (histidine)6-tagged pure protein (M r 28 000 or 43 000) inbinding buffer (Q B, 10% determination) or (histidine)6-tagged protein bound from E. coli extractColumn volume: 0.25 ml or 1 ml Flow rate: 0.25 ml/min or 1 ml/min Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4The Ni 2+-charged medium is compatible with all commonly used aqueous buffers, reducing agents, denaturants such as 6 M Gua-HCl and 8 M urea, and a range of other additives (see Table 2).Table 2. Ni Sepharose High Performance is compatible with the following compounds, at least at the concentrations given.1See Notes and blank run, p. 10–11.2Tested for 1 week at 40°C.3The strong chelator EDTA has been used successfully in some cases, at 1 mM.Generally, chelating agents should be used with caution (and only in the sample, not the buffers). Any metal-ion stripping may be counteracted by addition of a small excess of MgCl 2 before centrifugation/filtration of the sample. Note that stripping effects may vary with applied sample volume.Reducing agents 15 mM DTE 5 mM DTT20 mM ?-mercaptoethanol 5 mM TCEP10 mM reduced glutathione Denaturing agents 8 M urea 26 M guanidine hydrochloride 2Detergents2% Triton? X-100 (nonionic)2% Tween? 20 (nonionic)2% NP-40 (nonionic)2% cholate (anionic)1% CHAPS (zwitterionic)Other additives500 mM imidazole 20% ethanol 50% glycerol 100 mM Na 2SO 41.5 M NaCl 1 mM EDTA 360 mM citrate 3Buffer substances50 mM sodium phosphate, pH 7.4100 mM Tris-HCl, pH 7.4100 mM Tris-acetate, pH 7.4100 mM HEPES, pH 7.4100 mM MOPS, pH 7.4100 mM sodium acetate, pH 422General considerationsHisTrap HP is supplied precharged with Ni2+ ions. In general, Ni2+ is the preferred metal ion for purification of recombinant histidine-tagged proteins. Note, however, that in some cases it may be wise to test other metal ions, for example Zn2+ and Co2+, as the strength of binding depends on the nature of the histidine-tagged protein as well as the metal ion (see Optimization).We recommend binding at neutral to slightly alkaline pH (pH 7–8) in the presence of 0.5–1.0 M NaCl. Sodium phosphate buffers are often used. Tris-HCl can generally be used, but should be avoided in cases where the metal-protein affinity is very weak, since it may reduce binding strength. Avoid chelating agents such as EDTA or citrate in buffers, see Table2.Including salt, for example 0.5–1.0 M NaCl in the buffers andsamples, eliminates ion-exchange effects but can also have amarginal effect on the retention of proteins.Imidazole at low concentrations is commonly used in the binding and the wash buffers to minimize binding of host cell proteins. For the same reason, it is important to also include imidazole in thesample (generally, at the same concentration as in the washbuffer). At somewhat higher concentrations, imidazole may alsodecrease the binding of histidine-tagged proteins. The imidazole concentration must therefore be optimized to ensure the bestbalance of high purity (low binding of host cell proteins) and high yield (binding of histidine-tagged target protein). This optimalconcentration is different for different histidine-tagged proteins,and is usually slightly higher for Ni Sepharose High Performance than for similar IMAC media on the market (see Data File18-1174-40). Use highly pure imidazole; such imidazole givesessentially no absorbance at 280 nm.As alternatives to imidazole elution, histidine-tagged proteins can be eluted from the medium by several other methods orcombinations of methods – a lowering of pH within the range of2.5–7.5 can be used, for example. At pH values below 4, metal ionswill be stripped off the medium.Note:If the proteins are sensitive to low pH, we recommendcollection of the eluted fractions in tubes containing1 M Tris-HCl, pH 9.0 (60–200 µl/ml fraction) to restore thepH to neutral.Chelating agents such as EGTA or EDTA will also strip metal ionsfrom the medium and thereby cause protein elution, but the target protein pool will then contain Ni2+ ions. In this case, Ni2+ ions can be removed by desalting on a HiTrap? Desalting, a PD-10 Desalting Column, or HiPrep? 26/10 Desalting, (see Table3).Leakage of Ni2+ from Ni Sepharose High Performance is very low under all normal conditions, lower than for other IMAC mediatested. For applications where extremely low leakage duringpurification is critical, leakage can be even further reduced byperforming a blank run (see page 11). Likewise, a blank run should also be performed before applying buffers/ samples containingreducing agents (see page 11).Whatever conditions are chosen, HisTrap HP columns can beoperated with a syringe, peristaltic pump, or chromatographysystem.Note:If Peristaltic Pump P-1 is used, the maximum flow rate that can be run on a HisTrap HP 1 ml column is 3 ml/min.3OperationBuffer preparationWater and chemicals used for buffer preparation should be of high purity. Filter buffers through a 0.22 µm or a 0.45 µm filter beforeuse.Use high purity imidazole as this will give very low or noabsorbance at 280 nm.If the recombinant histidine-tagged protein is expressed asinclusion bodies, include 6 M Gua-HCl or 8 M urea in all buffers and sample. On-column refolding of the denatured protein may bepossible.Recommended conditionsSample preparationFor optimal growth, induction, and cell lysis conditions for your recombinant histidine-tagged clones, please refer to established protocols.Adjust the sample to the composition and pH of the binding buffer by: Adding buffer, NaCl, imidazole, and additives fromconcentrated stock solutions; by diluting the sample with binding buffer; or by buffer exchange, (see Table 3). Do not use strong bases or acids for pH-adjustment (precipitation risk). Filter the sample through a 0.22 µm or a 0.45 µm filter and/or centrifuge it immediately before applying it to the column.To prevent the binding of host cell proteins with exposed histidine, it is essential to include imidazole at a low concentration in the sample and binding buffer (see Optimization).Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl,20–40 mM imidazole, pH 7.4 (The optimal imidazole concentration is protein-dependent; 20–40 mM is suitable for many proteins.)Elution buffer:20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4 (The imidazole concentration required for elution is protein-dependent).Table 3. Prepacked columns for desalting and buffer exchangePurification1Fill the syringe or pump tubing with distilled water. Remove the stopper and connect the column to the syringe (use the luerconnector provided), laboratory pump or chromatographysystem tubing “drop-to-drop” to avoid introducing air into thesystem.2Remove the snap-off end at the column outlet.3Wash the column with 3–5 column volumes of distilled water.4Equilibrate the column with at least 5 column volumes of binding buffer. Recommended flow rates are 1 ml/min or5 ml/min for the 1 and 5 ml columns respectively.In some cases a blank run is recommended before finalequilibration/ sample application (see page 11).5Apply the pretreated sample using a syringe or a pump.6Wash with binding buffer until the absorbance reaches a steady baseline (generally, at least 10–15 column volumes).Note:Purification results are improved by using imidazole insample and binding buffer (see Optimization).7Elute with elution buffer using a one-step or linear gradient.Five column volumes are usually sufficient if the protein ofinterest is eluted by a one-step gradient. A shallow gradient, forexample a linear gradient over 20 column volumes or more,may separate proteins with similar binding strengths.Note:If imidazole needs to be removed from the protein, use HiTrap Desalting, a PD-10 Desalting Column, or HiPrep26/10 Desalting depending on the sample volume (seeTable3).Note:Ni Sepharose High Performance is compatible withreducing agents. However, removal of any weakly boundNi2+ ions by performing a blank run without reducingagents (as described on page 11) before applying buffer/sample including reducing agents is recommended. Do notleave HisTrap HP columns with buffers including reducingagents when not in use.Note:Leakage of Ni2+ from Ni Sepharose High Performance is low under all normal conditions. The leakage is lower than forother IMAC media tested (see Data File Ni Sepharose HighPerformance, 18-1174-40). For very critical applications,leakage during purification can be even further diminishedby performing a blank run (as described below) beforeloading sample.Blank run:Use binding buffer and elution buffer without reducing agents.1Wash the column with 5 column volumes of distilled water (to remove the 20% ethanol).2Wash with 5 column volumes of elution buffer.3Equilibrate with 10 column volumes of binding buffer.4OptimizationImidazole at low concentrations is commonly used in the binding and the wash buffers to minimize binding of host cell proteins. For the same reason, it is important to also include imidazole in thesample (generally, at the same concentration as in the washbuffer). At somewhat higher concentrations, imidazole may alsodecrease the binding of histidine-tagged proteins. The imidazole concentration must therefore be optimized to ensure the bestbalance of high purity (low binding of host cell proteins), and high yield (binding of histidine-tagged target protein). This optimalconcentration is different for different histidine-tagged proteins,and is usually slightly higher for Ni Sepharose High Performance than for similar IMAC media on the market (see Data File NiSepharose High Performance, 18-1174-40). Finding the optimalimidazole concentration for a specific histidine-tagged protein is a trial-and-error effort, but 20–40 mM in the binding and washbuffers is a good starting point for many proteins. Use a high purity imidazole, such imidazole gives essentially no absorbance at280 nm.Ni2+ is usually the first choice metal ion for purifying most(histidine)6-tagged recombinant proteins from nontagged host cell proteins, and also the ion most generally used. Nevertheless, it isnot always possible to predict which metal ion will be best for agiven protein. The strength of binding between a protein and ametal ion is affected by several factors, including the length,position, and exposure of the affinity tag on the protein, the type of ion used, and the pH of buffers, so some proteins may be easier to purify with ions other than Ni2+.A quick and efficient way to test this possibility and optimizeseparation conditions is to use HiTrap IMAC HP 1 ml columns,which are packed with IMAC Sepharose High Performance (notcharged with metal ions). Each column can be charged withdifferent metal ions, for example Cu2+, Co2+, Zn2+, Ca2+, or Fe2+.Instructions are included with each column.A study to compare the purification of six (histidine)6-taggedrecombinant proteins, including three variants of maltose-binding protein, with different metal ions has indicated that Ni2+ generally gives best selectivity between (histidine)-tagged and nontaggedhost-cell proteins (see Application Note 18-1145-18).5Stripping and rechargingNote:The column does not have to be stripped and recharged between each purification if the same protein is going to bepurified; it is sufficient to strip and recharge it after 5–7purifications, depending on the cell extract, extract volume,target protein, etc.Recommended stripping buffer: 20 mM sodium phosphate,0.5 M NaCl, 50 mM EDTA, pH 7.4Strip the column by washing with at least 5–10 column volumes of stripping buffer. Wash with at least 5–10 column volumes ofbinding buffer and 5–10 column volumes of distilled water before recharging the column.Recharge the water-washed column by loading 0.5 ml or 2.5 ml of0.1 M NiSO4 in distilled water on HisTrap HP 1 ml and 5 ml column,respectively. Salts of other metals, chlorides, or sulfates, may also be used (see“Optimization”). Wash with 5 column volumes distilled water, and 5 column volumes binding buffer (to adjust pH) before storage in 20% ethanol.6Cleaning-in-placeWhen an increase in back-pressure is seen, the column can becleaned. Before cleaning, strip off Ni2+ ions using therecommended procedure described above.After cleaning, store in 20% ethanol (wash with 5 column volumes) or recharge with Ni2+ prior to storage in ethanol.The Ni2+-stripped column can be cleaned by the followingmethods;Remove ionically bound proteins by washing the column with several column volumes of 1.5 M NaCl; then wash the columnwith approximately 10 column volumes of distilled water.Remove precipitated proteins, hydrophobically bound proteins, and lipoproteins by washing the column with 1 MNaOH, contact time usually 1–2 hours (12 hours or more forendotoxin removal). Then wash the column withapproximately 10 column volumes of binding buffer, followedby 5–10 column volumes of distilled water.Remove hydrophobically bound proteins, lipoproteins, and lipids by washing the column with 5–10 column volumes 30%isopropanol for about 15–20 minutes. Then wash the columnwith approximately 10 column volumes of distilled water.Alternatively, wash the column with 2 column volumes ofdetergent in a basic or acidic solution. Use, for example,0.1–0.5% nonionic detergent in 0.1 M acetic acid, contact time1–2 hours. After treatment, always remove residual detergentby washing with at least 5 column volumes of 70% ethanol.Then wash the column with approximately 10 column volumesof distilled water.7Scaling-upTwo or three HisTrap HP 1 ml or 5 ml columns can be connected in series for quick scale-up (note that back-pressure will increase).Ni Sepharose High Performance, the medium prepacked inHisTrap HP columns, is supplied preswollen in 25 and 100 ml labpacks (see Ordering Information). An alternative scale-up strategy is thus to pack the medium in empty columns – Tricorn? and XK columns are suitable for this purpose. 8StorageStore HisTrap HP columns in 20% ethanol at 4°C to 30°C.9TroubleshootingThe following tips may be of assistance. If you have any furtherquestions about your HisTrap HP column, please visit/doc/25f3fd1aa76e58fafab0030a.html /hitrap, contact our technical support, or your local representative.Note:When using high concentrations of urea or Gua-HCl,protein unfolding generally takes place. Refolding on-column (or after elution) is protein-dependent.Tips:To minimize dilution of the sample, solid urea or Gua-HCl can be added.Tips:Samples containing urea can be analyzed directly by SDS-PAGE whereas samples containing Gua-HCl must be buffer-exchangedto a buffer with urea before SDS-PAGE.Column has clogged:Cell debris in the sample may clog the column. Clean the column according to the section Cleaning-in-place.It is important to centrifuge and/or filter the sample through a0.22 µm or a 0.45 µm filter, see Sample preparation. Sample is too viscous:If the lysate is very viscous due to a high concentration of host nucleic acid, continue sonication until the viscosity is reduced,and/or add DNase I to 5 µg/ml, Mg2+ to 1 mM, and incubate onice for 10–15 min. Alternatively, draw the lysate through asyringe needle several times.Protein is difficult to dissolve or precipitates during purification:The following additives may be used: 2% Triton X-100, 2% Tween 20, 2% NP-40, 2% cholate, 1% CHAPS, 1.5 M NaCl,50% glycerol, 20 mM ?-mercaptoethanol, 1–3 mM DTT or DTE(up to 5 mM is possible but depends on the sample and thesample volume), 5 mM TCEP, 10 mM reduced glutathione, 8 Murea, or 6 M Gua-HCl. Mix gently for 30 min to aid solubilizationof the tagged protein (inclusion bodies may require longermixing). Note that Triton X-100 and NP-40 (but not Tween)have a high absorbance at 280 nm. Furthermore, detergentscannot be easily removed by buffer exchange.No histidine-tagged protein in the purified fractions:?Elution conditions are too mild (histidine-tagged protein still bound: Elute with an increasing imidazole gradient or decreasing pH to determine the optimal elution conditions.Protein has precipitated in the column: For the nextexperiment, decrease amount of sample, or decrease proteinconcentration by eluting with linear imidazole gradient insteadof imidazole steps. Try detergents or changed NaClconcentration, or elute under denaturing (unfolding)conditions (add 4–8 M urea or 4–6 M Gua-HCl).Nonspecific hydrophobic or other interaction: Add a nonionic detergent to the elution buffer (e.g. 0.2% Triton X-100)or increase the NaCl concentration.Concentration of imidazole in the sample and/or binding buffer is too high: The protein is found in the flow-through material. Decrease the imidazole concentration.Histidine-tag may be insufficiently exposed: The protein is found in the flowthrough material; perform purification of unfolded protein in urea or Gua-HCl as for inclusion bodies.Buffer/sample composition is incorrect: The protein is found in the flowthrough material. Check pH and composition of sample and binding buffer. Ensure that chelating or strongreducing agents are not present in the sample at too highconcentration, and that the concentration of imidazole is nottoo high.SDS-PAGE of samples collected during the preparation of thebacterial lysate may indicate that most of histidine-tagged protein is located in the centrifugation pellet. Possible causes and solutions are:Sonication may be insufficient: Cell disruption may be checked by microscopic examination or monitored bymeasuring the release of nucleic acids at A260. Addition oflysozyme (up to 0.1 volume of a 10 mg/ml lysozyme solution in25 mM Tris-HCl, pH 8.0) prior to sonication may improveresults. Avoid frothing and overheating as this may denaturethe target protein. Over-sonication can also lead tocopurification of host proteins with the target protein.The protein may be insoluble (inclusion bodies): The protein can usually be solubilized (and unfolded) from inclusion bodies using common denaturants such as 4–6 M Gua-HCl, 4–8 Murea, or strong detergents. Prepare buffers containing 20 mMsodium phosphate, 8 M urea, or 6 M Gua-HCl, and suitableimidazole concentrations, pH 7.4–7.6. Buffers with urea shouldalso include 500 mM NaCl. Use these buffers for samplepreparation, as binding buffer and as elution buffer. Forsample preparation and binding buffer, use 10–20 mMimidazole or the concentration selected during optimizationtrials (including urea or Gua-HCl).The protein is collected but is not pure (multiple bands on SDS polyacrylamide gel):Partial degradation of tagged protein by proteases: Add protease inhibitors (use EDTA with caution, see Table2). Contaminants have high affinity for nickel ions: Elute with a stepwise or linear imidazole gradient to determine optimal imidazole concentrations to use for binding and for wash; addimidazole to the sample in the same concentration as thebinding buffer. Wash before elution with binding buffercontaining as high concentration of imidazole as possible,without causing elution of the tagged protein. A shallowimidazole gradient (20 column volumes or more), mayseparate proteins with similar binding strengths. If optimizedconditions do not remove contaminants, further purificationby ion exchange chromotography (HiTrap Q HP orHiTrap SP HP) and/or gel filtration (Superdex? Peptide,Superdex 75 or Superdex 200) may be necessary.Contaminants are associated with tagged proteins: Add detergent and/or reducing agents before sonicating cells. Increase detergent levels (e.g. up to 2% Triton X-100 or2% Tween 20), or add glycerol (up to 50%) to the wash bufferto disrupt nonspecific interactions.Histidine-tagged protein is eluted during sample loading/wash:Buffer/sample composition is incorrect: Check pH and composition of sample and binding buffer. Ensure thatchelating or strong reducing agents are not present in thesample at a too high concentration, and that theconcentration of imidazole is not too high.Histidine-tag is partially obstructed: Purify under denaturing conditions (use 4–8 M urea or 4–6 M Gua-HCl).Column capacity is exceeded: Join two or three HisTrap HP1 ml columns together or change to a HisTrap HP 5 ml column. 10Intended useThe HisTrap HP is intended for research use only, and shall not be used in any clinical or in vitro procedures for diagnostic purposes. 11Ordering InformationProduct No. supplied Code No.HisTrap HP 5 × 1 ml 17-5247-01100 × 1 ml1 17-5247-051 × 5 ml 17-5248-015 × 5 ml 17-5248-02100 × 5 ml1 17-5248-05 Related products No. supplied Code No.100 ml 17-5268-02 HiTrap Desalting 5 × 5 ml 17-1408-01100 × 5 ml1 11-0003-29 PD-10 Desalting Column 30 17-0851-01 HiPrep 26/10 Desalting 1 × 53 ml 17-5087-014 × 53 ml 17-5087-02HisTrap FF 5 × 1 ml 17-5319-01100 × 1 ml1 17-5319-025 × 5 ml 17-5255-01100 × 5 ml1 17-5255-02 HisTrap FF crude 5 × 1 ml 11-0004-58100 × 1 ml1 11-0004-595 × 5 ml 17-5286-01100 × 5 ml1 17-5286-02 HisTrap FF crude Kit 1 kit 28-4014-77 HisPrep? FF 16/10 1 × 20 ml 28-9365-511Pack size available by special order.1 One connector included in each HiTrap package.2 Two, five, or seven stop plugs female included in HiTrap packages depending on the product.3One fingertight stop plug is connected to the top of each HiTrap column at delivery.AccessoriesNo. Supplied Code No.1Tubing connector flangeless/M6 female 12 18-1003-68Tubing connector flangeless/M6 male 1 2 18-1017-98Union 1/16” female/M6 male 1 6 18-1112-57Union M6 female /1/16” male 1 5 18-3858-01Union luerlock female/M6 female 218-1027-12HiTrap/HiPrep, 1/16” male connector for ?KTA design8 28-4010-81Stop plug female, 1/16”2 5 11-0004-64Fingertight stop plug, 1/16”35 11-0003-55Related literatureCode No.Recombinant Protein Purification Handbook, Principles and Methods18-1142-75Affinity Chromatography Handbook, Principles and Methods18-1022-29Affinity Chromatography, Columns and Media Selection Guide。

海报设计模板英语作文Poster Design Template English Composition。

Poster design is an essential part of marketing and communication. It is a visual representation of an idea, message, or event that aims to attract attention and convey information. Creating an effective poster design requires careful consideration of various elements such as layout, typography, color, and imagery. In this article, we will discuss the key components of a poster design template in English composition.1. Title and Headline。

The title and headline are the first things that viewers will notice on a poster. It should be clear, concise, and attention-grabbing. The title should communicate the main message of the poster, while the headline can provide additional information or a call to action. The font size and style should be chosen carefully to ensure readability from a distance.2. Imagery。