GST gene fusion system-GE Healthcare

GE HealthcareBedside flexibility and adaptabilityThe Dash® monitoring family is a portable monitoring system that is flexible and easy to use. Dash allows the acuity of any bed to be modified to meet changing patient needs.Gold standard algorithms and technologyDash monitors revolutionize patient care and assessment by combining the most complete selection of gold standard patient monitoring parameters with leading-edge cardiac technology.Enterprise networkingHard-wired and wireless network connectivity – including access to CIS, CVIS, PACS, RIS, HIS and more than 350 beds without central station support – contributes to the Dash monitors’ unprecedented ability to adapt to changing patient acuity demands.Dash 3000, 4000 & 5000 Flexible acuity monitoringSize Dash 3000 – 8.4 in., Dash 4000 – 10.4 in., Dash 5000 – 12.1 in.Type Active-matrix color LCDResolution Dash 3000 and 4000: 640 by 480 dpi, Dash 5000: 800 by 600 dpiNumber of traces 7 (maximum)Number of seconds/trace 4.9 at 25 mm/secSweep speed 6.25, 12.5, 25 mm/sec (with erase bar)TrimKnob® controlFive hard keys Standard Silence Alarm, Print, NBP Go/Stop, Zero All and Power On/Off. Dash 5000 addsTrends, NBP Auto, Admit/Discharge, Standby and Main ViewRemote control option AvailableCategories Patient status and system statusPriority 4 levels – Crisis, Warning, Advisory and MessageNotification Audible and visualSetting Default and individualSilencing 1 minute, current alarm onlyPause 5 minutes in Adult ICU mode, 3 minutes in Neonatal ICU mode and 5 minute,15 minute, or permanent pause in OR modeVolume Default 70 dB measured at 1 meterNumber of channels 1 to 4 (optional)Transducer sites Arterial, femoral artery, pulmonary arterial, central venous, right atrial, left atrial,intracranial and specialTransducer requirements Excitation voltage: 5 V dc ± 0.1%Transducer output 5 µV/V/mmHgRange -25 mmHg to 300 mmHgOffset ± 150 mmHgFrequency response dc to 50 Hz (-3 dB)Zero balance range ± 150 mmHgZero balance accuracy ± 1 mmHgZero balance drift ± 1 mmHg over 24 hoursAccuracy ± 2% or ± 1 mmHg, whichever is greater(exclusive of transducer)Alarms User-selectable upper and lower limits for systolic, diastolic and mean pressuresStandard leads available I, II, III, V, aVR, aVL and aVF3 leadwire I, II, or III5 leadwire I, II, III, V, AVR, AVL, and AVF10 leadwire I, II, III, AVR, AVL, AVF, VI, V2, V3, V4, V5 and V6Leads analyzed simultaneously I, II, III and V (multi-lead mode)Lead fail Identifies failed leadAlarms User-selectable upper and lower heart rate limitsVoltage range ± 0.5 mV to ± 5 mVSignal width 40 ms to 120 ms (Q to S)Heart rate range 30 to 300 bpmInput impedance Common mode > 10 MΩ at 50/60 HzDifferential > 2.5 MΩ from dc to 60 HzCommon mode rejection 90 dB minimum at 50 or 60 HzImpulse response For an impulse of 3 mV applied for 100 msDisplacement following impulse < 0.1 mVSlope following impulse < 0.3 mV/sFrequency response R esponse of non-permanent displays is limited by resolution to 40 Hz (-3 dB)@25 mm/s. Specified upper frequency limits may vary by ± 2 Hz.Diagnostic mode 0.67 Hz (+0.4 dB) to 100 Hz (-3 dB)For compliance with China 1.0 Hz (+0.4dB) to 75 Hz (-3 dB)National StandardMonitoring mode 0.67 (+0.4 dB) to 40 Hz (-3 dB)Moderate mode 0.67 (+0.4 dB) to 25 Hz (-3 dB)Maximum mode 5.0 Hz (-0.3 dB) to 25 Hz (-3 dB)Noise < 30 µV (referred to input)Input voltage range ± 2 mV to ± 700 mVInput pulse width 0.1 ms to 2 msRise time 10 µs to 100 µsOver/under shoot 2 mV (max)Baseline drift < 0.5 mV per hour with a ± 700 mV, 2 msPacemaker pulse AppliedMeasurement technique Impedance variation detectionRange 0-200 breaths per minute for variations of 1.0 – 10.0 ΩRespiration rate 0-200 breaths per minuteBase impedance 100-1000 Ω at 52.6 kHzDetection sensitivity 0.4 to 10 Ω variationWaveform display bandwidth 0.1 to 1.8 Hz (-3 dB)Alarms User-selectable upper and lower respiration rate limits, and user-selectable apnea limitNumber of channels 2Probe type YSI Series 400 or 700 (determined by input cable)Temperature range 0°C to 45°C (32°F to 113°F)Resolution ± 0.1°CParameters displayed T1, T2Accuracy (independent of source) ± 0.1°C for YSI series 400; ± 0.3°C for YSI series 700 probes Alarms User-selectable upper and lower limits for T1, T2Probe type In-line or bath probeCatheter size 5F, 6F, 7F, 7.5F and 8FInjectate volume 3, 5 or 10 ccParameters displayed Cardiac output, blood temperature, injectate temperature and trial numberCardiac output 0.2-15 (liters per minute)Blood temperature 30-42°CInjectate temperature 0-30°CCardiac output ± 5%Blood temperature ± 0.2°CInjectate temperature ± 0.3°CFrequency response dc to 15 Hz ± 2 HzParameters monitored Arterial oxygen saturation (SpO2) and peripheral pulse rate (PPR)SpO2 range Nellcor 1-100%; Masimo 30-100%; GE Ohmeda 30-100%PPR range Nellcor 20-300 BPM; Masimo 25-240 BPM; GE Ohmeda 30-250Accuracy Actual accuracy depends on probe. Please referencemanufacturer’s specifications.Nellcor SpO2 ± 2 digits (70-100% SpO2)Masimo SpO2 ± 2% Adults/Pediatric (70-100% SpO2)GE Ohmeda SpO2 ± 2% (70-100% SpO2), SpO2 ± 3% Neonates, ≤ 69% unspecifiedPPR ± 3 beats per minuteAlarms User-selectable upper and lower limits for SpO2 and PPRTechnology DINAMAP® classic and SuperSTAT™ (SuperSTAT onlyavailable with Masimo and Nellcor SpO2)Measurement technique OscillometricDisplayed parameters Systolic, diastolic and mean pressures, time oflast measurementMeasurement modes Adult ICU and OR modes; manual, auto and stat,neonatal mode; manual and autoAdult 30-285 mmHgPediatric 30-235 mmHgNeonate 30-140 mmHgAdult 20-260 mmHgPediatric 20-220 mmHgNeonate 20-125 mmHgAdult 10-220 mmHgPediatric 10-210 mmHgNeonate 10-110 mmHgAdult 30-200 bpmPediatric 30-200 bpmNeonate 30-200 bpmOverall system accuracy Meets or exceeds SP 10-1992 AAMI standards Automatic cycle times 0-4 hoursTubing length 12 feet adult, 8 feet neonatalAutomatic cuff deflation Cycle time exceeding 3 minutes (90 seconds neonatal),French mode – Cycle time exceeding 2 minutes (60 secondsneonatal), power off, or cuff pressure exceeds 294 mmHg(± 6 mmHg) adult, 250 (± 5 mmHg) pediatric,147 (± 3 mmHg) neonatalCuff sizes Thigh, large adult, adult, small adult, child, infant andneonatal, sizes #5 - #1 and assorted long sizesAlarms User-selectable upper and lower limits for systolic,diastolic and mean pressures2Supports Novametrix CapnoStat (mainstream) and CapnoFlex LF (low-flow sidestream) CO2 technologiesPrinciple of operation Non-dispersive infrared (NDIR) single beam optics,dual wavelength and no moving partsWarm-up time 2 minutes warm-up time to meet accuracy specifications; waveform immediateupon power up, calculated end tidal after two breathsCable length (mainstream) 8 foot (2.4 m)Sample line length (low-flow sidestream) 7 foot (2.1 m)Inspired and expired CO2 concentrations in %, mmHg or kPa; respiratory rate, continuous CO2 waveform0-100 mmHg, 0-13%, 0-12.5 kPaPiCO2 /FiCO20-50 mmHg, 0-6.5%, 0-6.25 kPaRespiration rate range Low-flow SS 0-150 breaths/minMainstream 0-120 breaths/minMS ±2 mmHg or 5%, whichever is greaterSS 0-40 mmHg ± 2 mmHg; 41-70 mmHg ± 5% of reading;71-100 mmHg ± 8% of reading; all specifications ± 12%of actual from 80-150 BrPMDisplay resolution 1 mmHgRise time Less than 200 ms (low-flow sidestream); less than 60 ms(mainstream adult reusable or SPU); less than 50 ms(mainstream infant reusable or SPU)Respiration rate accuracy ± 1 breath/minAutomatic barometric pressure ± 25 mmHg from 530-785 mmHgOperator-selectable O2/N2O compensationMainstream No routine user calibration required. 15 second airway adapter zero performedwhen changing to a different style of airway adapter.Low-flow sidestream No routine user calibration requiredTypes Adult reusable (standard), adult disposable, infantDeadspace Adult reusable/disposable < 5 ccInfant disposable < 1 ccTaper meets ISO 5356-1Types Adult reusable (standard), adult disposable, infantDeadspace Adult reusable/disposable < 7.3 cc infant disposable < 1 ccAdult, pediatric and infant Nasal CO2 and nasal CO2/O2A dult and pediatric Nasal/oral CO2 and nasal/oral CO2/O2CO2High inspired CO2; high/low expired CO2Respiratory rate Adjustable high and lowMethod Thermal dot arrayHorizontal resolution 480 dots/in. @25 mm/sec.Vertical resolution 200 dots/in.Number of waveform channels fourPaper width 50 mm (1.97 in.)Paper length 30 m (100 ft.)Paper speed 0.1, 0.5, 1, 5, 10, 12.5, 25 and 50 mm/sec. (± 2%)Gain 1 V/mV ± 10%DC offset ± 100 mV (max)Noise < 5 mV peak to peak 0-300 HzFrequency response Refer to frequency response section under ECGGain 10 mV/mmHg ± 2%DC offset ± 20 mV (max)Noise < 5 mV peak to peak 0-300 HzFrequency response dc to 50 Hz-0/+2 HzOperating frequency 2.4 to 2.5 GHzTransmit power 100 mWData rate (802.11) 1Mbps and 2Mbps per channel; (802.11b) 1, 2, 5.5, 11 MbpsRadio technology Frequency-hopping spread spectrumCommunication protocol IEEE 802.11 or IEEEE 802.11bUL 1950 Listed (ITE 9B97), CE Mark RF StandardUS/CAN FCC Part 15 Class B, RSS-210, Europe: ETSI EN 300 328, Japan: RCD STD-33RBattery type Exchangeable Lithium-IonMaximum number of batteries 2Voltage 11.1 V (nominal)Capacity ≥ 3.45 Ah (varies with manufacturers)Charge time Less than 4 hours eachRun time 4 to 5 hoursBattery life 500 cycles to 50% capacityPower requirements 90-132 VAC 50/60 Hz 2.0A190-264 VAC 50/60 Hz 1.0APower consumption 75 W (fully loaded)Cooling ConvectionHeat dissipation 240 Btu/hr. (maxGE imagination at workGE HealthcareP.O. Box 900, FIN-00031 GE, FinlandTel. +358 10 394 11 • Fax +358 9 146 ©2006 General Electric Company – All rights reserved.GE and GE Monogram are trademarks of General Electric Company.Masimo and SET are registered trademarks of Masimo Corporation.Nellcor is a registered trademark of Nellcor Puritan Bennett, Inc.Dash, TrimKnob, DINAMAP and SuperSTAT are registered trademarks of General Electric Company.GE Healthcare Oy., doing business as GE Healthcare.CertificationUL2601-1 classifiedIEC 60601-1 certifiedCE Marking for the 93/42/EEC UL Classified for CAN/CSA Medical Device Directive C22.2 No. 601.12028034-002-2006.03-V2.0Do not exceed Maximum 70°C (158°F) at 95% relative humidity Minimum -40°C (-40°F)CO 2 sensor -30 to 65°C (-22 to 149°F)Batteries-20 to 60°C (-4 to 140°F)Ambient temperature 0-40°C (32-104°F) (Nellcor 0-35°C (32-95°F))While charging batteries 0-35°C (35-95°F)CO 2 sensor 10-40°C (50-104°F)Relative humidity 5-95% @40°CVibration MIL-STD 810E, Method 514.4, Category 1Altitude-273 m to 2,943 m (-896 to 9,655 ft.)WarrantyStandard warranty is one year.。

一种高亲和结合I型胶原蛋白的GST融合蛋白表达分析董育红;李遂焰;陈渝萍【摘要】鉴于纤维化疾病病人组织中I型胶原的异常沉积和人胎盘生长因子2氨基酸123～144肽段(PlGF-2123-144)对I型胶原蛋白出众的高亲性,尝试制备PlGF-2123-144短肽用于改善抗纤维化药物的组织靶向特异性.首先对该短肽的编码DNA序列进行修改和扩增,构建了GST-PlGF-2123-144融合蛋白的原核表达质粒;进而使用E.coli BL21诱导表达该融合蛋白,并以谷胱甘肽琼脂糖纯化后透析;最后采用ELISA检测确定,经原核表达和纯化所获得的GST-PlGF-2123-14融合蛋白具备对I型胶原蛋白的高亲性.为研发特异性靶向纤维化组织的抗纤维化药物打下了可靠基础.【期刊名称】《生物学杂志》【年(卷),期】2019(036)005【总页数】4页(P21-24)【关键词】I型胶原蛋白;PlGF-2123-144;GST融合蛋白;原核表达【作者】董育红;李遂焰;陈渝萍【作者单位】西南交通大学生命科学与工程学院,成都610031;西南交通大学生命科学与工程学院,成都610031;西南交通大学材料科学与工程学院,成都610031;南华大学药学与生命科学学院,衡阳421001【正文语种】中文【中图分类】Q78当前临床治疗亟待解决的难题是如何提高药物治疗疗效并降低乃至去除其毒副作用，药物的靶向性研究越来越受到重视[1-2]。

纤维化疾病(包括系统性硬化、肺纤维化、肝硬化、进展性肾病、骨髓纤维化等)病人的组织中异常沉积细胞外基质蛋白(ECM)，使组织变硬、顺应性降低，破坏组织器官的结构和功能，危害人们的健康乃至生命[3-7]。

其中，I型胶原蛋白的增多极为显著[8-12]。

人类胎盘生长因子2(Placental Growth Factor，PlGF-2)的氨基酸123～144肽段(PlGF-2123-144)介导其对多种ECM蛋白尤其是对I型胶原蛋白的高亲性[13]。

GE HealthcarepGEX-4T-3GST Expression VectorProduct Specification SheetCode: 28-9545-52WarningFor research use only.Not recommended or intended for diagnosis of disease in humans or animals.Do not use internally or externally in humans or animals. HandlingThe vector should be removed from the dri-ice packaging and stored at -20°C. After thawing, centrifuge briefly to recover contents.ExpiryVector is stable for a minimum of 8 weeks from date of receipt when stored under recommended storage conditions.Safety warnings and precautionsAll chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet(s) and/or safety statement(s) for specific advice. Components25 µg vector supplied in 10 mM Tris, 1 mM EDTA pH 8.0. Quality controlPurified plasmid will contain predominantly supercoiled format typically greater than 90% by agarose gel electrophoresis. Chromasomal DNA from the host is not observed. Plasmid is assayed to demonstrate presence of B am H1; Eco R I; Not I restriction endonuclease sites.ProtocolsPrepare fusion construct by inserting gene of interest into the multiple cloning site of pGEX-4T-3 using any one, or combination of unique restriction sites and transform into a host of choice such as E. coli BL21 (27-1542-01).Growth and Induction:1.Dilute an overnight culture transformed with pGEX fusionconstruct, 1:10 in fresh complex medium containing 100 µg/ml ampicillin. Grow the cells at 37°C to mid-log phase(A600 = 0.6–1.0). 2.Induce expression of fusion proteins by adding isopropyl-βD-thiogalactoside (IPTG) to 0.1 mM final concentration and allow the cells to grow for an additional 3–5 hours at 37°C.3.Expression of GST fusion proteins can be monitored using theAnti-GST Antibody (27-4577-01), GST Detection Modules(27-4590-01, 27-4592-01) or ECL GST Western Blotting Detection Kit (RPN1237).Preparation of cell extracts:1.Sediment the cells by centrifugation and resuspend in 1/20volume of PBS (PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,1.8 mM KH2PO4, pH 7.3).2.Lyse the cells by mild sonication or chemical lysis.3.Add Triton X-100 to a final concentration of 1% and mix gently atroom temperature (25°C) for 30 minutes to solubilize proteins. 4.Centrifuge the crude extract at 10 000 × g for 5 minutes at 4°C. PurificationThere are a range of Gluthatione Sepharose™ prepacked column and bulk media products available to purify GST Fusion proteins. For manual purification of sample volumes up to 600 µl use GST SpinTrap™ microspin columns or GST MultiTrap™ 4B 96-well plates. For sample volumes between 600 µl and 10 ml use GST GraviTrap™ gravity flow column. Where sample volumes are above 10 ml,use LabMate™ reservoir together with GST GraviTrap. All formats described can be used for preparation of samples in parallel. In addition GST HiTrap™ 1 and 5 ml columns and GST HiPrep™ FF16/10 column are available for purification in a chromatography system such as the ÄKTA™ design system. Alternatively, Gluthatione Sepharose bulk media are available from 10 ml up to 500 ml. A GST Bulk Kit is also available combining 10 ml Gluthatione Sepharose4B bulk medium and 5 empty columns with required buffers. For simplified buffer preparation use the GST Buffer Kit. Ordering information for all associated products is listed below.Site-specific proteolysis of fusion proteins:Separation of the recombinant protein from the glutathioneS-transferase moiety may be accomplished by site specific proteolysis using bovine thrombin (27-0846-01). Exact reaction conditions will vary with the nature of the fusion protein. The following conditions may be used as a guideline and should be optimized for each fusion protein: thrombin concentration, 0.2% (w/w) of fusion protein; reaction buffer, PBS; incubation temperature, 25°C; incubation time, 2–16 hours (1, 2).Multiple Cloning region and protease cleavage siteFor more information on the use of pGEX vectors, see GST Gene Fusion System Handbook.Intracellular expression of some eukaryotic proteins in Escherichia coli can lead to the formation of inclusion bodies (2). Increased solubilities can be obtained by lowering the growth temperature from 37°C to 28–30°C (3). Shortening the induction period may also improve results. Exact conditions must be established for each protein.imagination at workThe following primers for double-stranded sequencing of pGEX vectors are available: 5’ pGEX Sequencing Primer (bases 869–891)and 3’ pGEX Sequencing Primer (bases 1020-998).Further information relating to DNA sequence, restriction maps and control regions can be found at: References1. Smith, D. B. and Johnson, K. S., Gene 67, 31 (1988).2. Eaton, D., et al., Biochemistry 25, 505 (1986).3. Schein, C. H. and Noteborn, M. H. M., Bio/Technology 6, 291 (1988).4. Smith, D. B. and Corcoran, L. M., Current Protocols , pg. 16.7.1 (1990).Related productsGST vector products Code No. pGEX-4T-2 (25 µg) 28-9545-50pGEX-5X-1 (25 µg) 28-9545-53 pGEX-5X-2 (25 µg) 28-9545-54 pGEX-5X-3 (25 µg) 28-9545-55 pGEX-2TK (25 µg) 28-9546-46 pGEX-6P-1 (25 µg) 28-9546-48 pGEX-6P-2 (25 µg) 28-9546-50 pGEX-6P-3 (25 µg) 28-9546-51 pGEX-2T (25 µg)28-9546-53 pGEX-1λT EcoR/BAP (5 µg)28-9546-56pGEX 5’ Sequencing Primer5’-d[GGG-CTGGCAAGCCACGTTTGGTG]-3´ 27-1410-01 pGEX 3’ Sequencing Primer 5’-d[CCG-GGAGCTGCATGTGTCAGAGG]-3’ 27-1411-01 E. coli BL21 1 vial27-1542-01 GST purification products Code No. GST GraviTrap (10 columns) 28-9523-60 LabMate PD-10 Buffer Reservoir (50) 18-3216-03 GST Buffer Kit 28-9523-61 GST Bulk Kit27-4570-01 GST SpinTrap (50 columns)28-9523-59 GST MultiTrap 4B (4 × 96-well plates) 28-4055-00 GST MultiTrap 4 FF (4 × 96-well plates) 28-4055-01 GSTrap 4B (5 × 1 ml) 28-4017-45GSTrap 4B (100 × 1 ml)1 28-4017-46GSTrap 4B (1 × 5 ml ) 28-4017-47GSTrap 4B (5 × 5 ml) 28-4017-48GSTrap 4B (100 × 5 ml)1 28-4017-49Glutathione Sepharose 4B (10 ml) 17-0756-01Glutathione Sepharose 4B (100 ml) 17-0756-05Glutathione Sepharose 4B (300 ml)17-0756-041 Pack size available by specific customer order.GST detection product Code No. GST Detection Module 27-4590-01GST Detection Module (96-well format) 27-4592-01 Anti-GST Antibody27-4577-01 ECL GST Western Blotting Detection Kit RPN1237 Site-specific ProteasesCode No. PreScission Protease (500 units) 27-0843-01Thrombin (500 units) 27-0846-01Factor Xa (400 units)27-0849-01Lysis kitCode No.Yeast Protein Extraction Buffer Kit 28-9440-45Mammalian Protein Extraction Buffer 28-9412-79LiteratureCode No.GST Gene Fusion System Handbook 18-1157-58Recombinant Protein Purification Handbook 18-1142-75Affinity Chromatography Handbook18-1022-29LegalGE, imagination at work and GE Monogram are trademarks of General Electric Company.ÄKTA, ECL, GraviTrap, HiTrap, HiPrep, LabMate, MultiTrap, Sepharose and SpinTrap are trademarks of GE Healthcare companies. pGEX vectors: pGEX vectors are to be used for scientificinvestigation and research and for no other purpose whatsoever and a license for commercial use of the licensed products and processes claimed in US patent 5,654,176 and equivalent patents and patent applications in other countries must be negotiated directly with Millipore Corp (formely Chemicon International Inc) by the purchaser prior to use.All third party trademarks are the property of their respective owners.© 2009 General Electric Company – All rights reserved. First published June 2009All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information.For your local office contact information, visit /contact GE Healthcare UK Limited Amersham PlaceLittle Chalfont, Buckinghamshire, HP7 9NA, UK GE Healthcare offices:GE Healthcare Bio-Sciences AB Björkgatan 30, 751 84 Uppsala, SwedenGE Healthcare Europe GmbHMunzinger Strasse 5, D-79111 Freiburg, GermanyGE Healthcare Bio-Sciences Corp.800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327, USAGE Healthcare Bio-Sciences KK Sanken Bldg. 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan2895958628-9545-52PS AA 06/2009。

GST标签的去除GST标签的去除可在目的蛋白的功能或结构研究之前进行。

根据目的蛋白的性质不同，完全消化所需的蛋白酶量、温度、及孵育长度也各异。

含有PreScission蛋白酶、凝血酶或Xa因子识别位点的标签蛋白可在与Glutathione Sepharose 层析填料结合时切割，或者洗脱后在溶液中切割。

当GST蛋白与柱结合时，切割释放了目的蛋白，它与结合缓冲液一起洗脱，而GST部分仍与填料结合。

一般来说，柱上切割是推荐使用的方法，因为许多潜在的杂质都能洗掉，目的蛋白在洗脱时具有更高的纯度。

如果需要优化切割条件，建议洗脱后切割。

从蛋白或肽段制备中去除丝氨酸蛋白酶如凝血酶、Xa因子和PreScission蛋白酶，可利用Benzamidine Sepharose 4 Fast Flow来进行，此填料有大包装。

为了方便，Benzamidine Sepharose 4 Fast Flow也有预填充的HiTrap Benzamidine FF 1 ml和5 ml柱。

凝血酶凝血酶能够对带有可接近的凝血酶识别序列的融合蛋白进行位点特异的切割，用于消化包含凝血酶识别序列的pGEX载体所制备的GST标签蛋白（pGEX-1λT、pGEX-2T、pGEX-2TK、pGEX-4T-1、pGEX-4T-2及pGEX-4T-3）。

在22°C，1×PBS中，一个单位的酶16小时可切割100 μg待测GST标签蛋白，切割效率在90%以上。

Xa因子纯化自牛血浆的Xa因子特异切割四肽Ile-Glu-Gly-Arg之后的蛋白，可用于消化包含此序列的pGEX载体所制备的GST标签蛋白（pGEX-3X、pGEX-5X-1、pGEX-5X-2和pGEX-5X-3）。

在22°C，1 mM CaCl2、100 mM NaCl和50 mM Tris-HCl（pH 8.0）的反应体系中，一个单位的酶16小时可切割100 μg待测GST标签蛋白，切割效率在90%以上。

Installation GuideInstallation Kit for Mounting Philips MP90 on GE Healthcare Aisys CarestationThe purpose of this guide is to: 1. Describe attachment of Top Plate to machine.2. Describe attachment of Display Mounting Bracket to display arm.3. Describe mounting of displays on Display Mounting Bracket.4. Describe attachment of Down Post to Articulating Arm.5. Describe mounting of Articulating Arm on left side of anesthesia machine.Attaching Top Plate to Anesthesia MachineThe following parts and hardware are included with this installation kit (hardware not shown):Tools required: Phillips screwdriver (not provided).1. Locate the four (4) threaded inserts shown in photo below left. Installation Notes: It may be necessary to remove hole covers to expose the threaded inserts. The right front mounting hole will be covered by the front Camlock Rail (see step 3).2. Align three (3) mounting holes in Top Plate with corresponding threaded inserts in top of machine. Fasten Top Plate to machine with three (3) M5 x 18mm FHMS. Installation Note: A fourth screw will be installed in step3.Item # DescriptionQty 1 Top Plate/Camlock Rail Assembly1 2M5 x 12mm Flat Head Machine Screw (FHMS)4Fasten Top Plate to Machine with M5 x 18mm FHMS (3) Threaded Inserts (4)3. Unscrew and remove right and center screws from front Camlock Rail. Loosen left screw and rotate Rail enough toallow access to fourth mounting hole. Install M5 x 18mm FHMS in threaded insert. Return Rail to original mounting position, reinstall right and center screws, and tighten left screw in Camlock Rail.Remove Right & Center ScrewsLoosen Left ScrewM5 x 18mm FHMS (1)Attaching the Display Mounting Bracket to the Display ArmThe following parts and hardware are included with this installation kit (hardware not shown):Tools Required: 3/16'' hex wrench (provided), Phillips screwdriver (not provided).1. Remove existing display and counterweight from display arm in accordance with the Aisys Technical Reference Manual.Item # DescriptionQty1Display Mounting Bracket1 2 Remote Keypad Bracket 13 M6 x 16mm Socket Head Cap Screw (SHCS)4 4M4 x 12mm Pan Head Machine Screw (PHMS)85 3/16'' Hex Wrench1 Display Arm with Display and Counterweight Removed2. Align Display Mounting Bracket with mounting holes in display arm. Installation Note: Ensure Display Mounting Bracket is angled away from the arm. Using the 3/16'' hex wrench provided, fasten Mounting Adapter to display arm with four (4) M6 x 16mm SHCS.Mounting a Display, Remote Speedpoint, External Alert Device, and Power SupplySeveral display-mounting options are provided in this installation kit. Refer to the following separate installation guides for mounting procedures and application information. Please read the following installation notes before mounting a display.Installation Notes1. Mounting Kit for 75/100 VESA Compatible Flat Panel Displays (DU-FLP-0002-17): This installation kit may be required to convert a 100 x 100mm mounting pattern to a 75 x 75mm mounting pattern. This kit contains screws and spacers which may be required for mounting the flat panel display.2. Mounting an M8033C (17'') or M8031B (15'') display: A Spacer (provided) is required for mounting either display. Insert appropriate Spacer and mount display as shown on pages 6 - 7.17'' display : Use M4 pan head machine screws plus VESA Mounting Adapter from Mounting Kit for 75/100 VESA Compatible Flat Panel Displays.15'' display: Use M4 pan head machine screws from Mounting Kit for 75/100 VESA Compatible Flat Panel Displays.3. Philips MP90 Remote Speedpoint Mounting Bracket and Adapter, and External Alert Device Mounting Bracket (AG-0019-80). This installation kit includes a separate installation guide (DU-AG-0019-80) which should be used in conjunction with pages 6-7 for mounting remote speedpoint bracket, external alert device and power supply.M6 x 16mm SHCS (4)Display Mounting BracketMounting Holes (4)Mounting a Display on the Display Mounting BracketInstallation Note: Display Mounting Bracket shown detached from display arm for clarification.17'' (M8033C) Display Mount display as shown below.15'' (M8031B) DisplayMount display as shown below.M4 x 20mm PHMS (2)SpacerAlarm BracketMounting a 12'' Display on the Display Mounting BracketMount display as shown below.Thread M4 x 12mm PHMS (4)into Rear of DisplayAttaching Down Post to Articulating Arm and Mounting Arm in Left ChannelThe following parts and hardware are included with this installation kit (hardware not shown):Tools required: 5/32'' and 1/8'' hex wrenches (provided).1. Using the 5/32'' hex wrench provided, fasten Post to Swivel Cup with three (3) #10-32 x 3/8'' SHCS as shown below.2. Insert Slide (rear of Arm) in bottom of channel and move to desired mounting position. Using the 1/8'' hex wrench provided, tighten two (2) socket head set screws at bottom of Slide.Item # DescriptionQty 1 M-Series Articulating Arm, 12'' x 12'' 1 2 Down Post, 6''1 3#10-32 x 3/8'' Socket Head Cap Screw (SHCS)3 4 5/32'' Hex Wrench 1 5 1/8'' Hex Wrench1M-Series Arm#10-32 x 3/8'' SHCS (3)6'' Post Swivel Cup 1Insert Slide Tighten Set ScrewsBottom of SlideMounting Philips MP90 Equipment on Camlock Rails & Down Post1. Mount EGM and CPU on camlock rails according to Philips instructions.2. Follow Philips instructions for mounting FMS on Down Post. A Philips-supplied clamp is required for this installation.EGM and CPU in Camlock Rails FMS Clamped to Down Post。

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。

摘要：用AKTAxpressTM智能蛋白质多维纯化系统进行两步、全自动纯化，从E . C o l i 裂解液中纯化G S T - 标记的hippocalcin (海马钙结合蛋白)。

两步法的第一步是亲和层析捕获，该步主要使用1 ml GSTrap TM 4B柱，第二步是凝胶过滤，该步主要使用HiLoadTM 16/60 SuperdexTM200 pg 柱。

使用两步纯化法可以获得高纯度的SThippocalcin，并显示出GST-hippocalcin目标蛋白质的二聚体和一个大的多聚体。

用AKTAxpress 色谱系统GSTrap 4B 柱和Hiload 16/60 Superdex200 pg 柱进行两步法全自动纯化GST-hippocalcinPerkinElmer微孔板系列产品现货火热促销中，咨询相关情况>> >>J. Lundqvist and A. KarlssonGE Healthcare, Uppsala, Sweden用AKTAxpressTM智能蛋白质多维纯化系统进行两步、全自动纯化，从E . C o l i 裂解液中纯化G S T - 标记的hippocalcin (海马钙结合蛋白)。

两步法的第一步是亲和层析捕获，该步主要使用1 ml GSTrap TM 4B柱，第二步是凝胶过滤，该步主要使用HiLoadTM 16/60 SuperdexTM200 pg 柱。

使用两步纯化法可以获得高纯度的SThippocalcin，并显示出GST-hippocalcin目标蛋白质的二聚体和一个大的多聚体。

导言谷胱甘肽S- 转移酶 (GST) 是继组氨酸之后的第二个应用最多的重组蛋白标记。

GST 基因融合系统利用质粒pGEX在E.coli中进行可诱导、高水平细胞内表达基因或与日本血吸虫 (Schistoma japonicum) GST 融合的基因片段 (1)。

通过用谷胱苷肽琼脂糖的层析柱捕获，然后用还原型谷胱苷肽洗脱可以很方便地纯化出标记蛋白质。

Getting the best out of your Biacore™ system Biacore consumablesA flying startNo matter what you want to get out of your interaction analysis, GE Healthcare has developed a range of tools designed specifically to make Biacore assays as easy and reliable as possible.The complete toolbox is backed up by stringent production methods and quality control.This brochure describes some of the benefits of Biacore consumables for rapidly getting great results. We’ve labeled tools with colors to indicate the application areas where the tool offers clear advantages.Give yourself a flying start to successful Biacore analysis, and land on reliable results.A sensor surface for every needGet the best response for your particular interaction study Kits to save you time and effortUp and running in record time with our kits and reagents Buffers and solutions for convenienceGet a quick start and feel confident in your daily workProducts marked with thesesymbols are great for:Biotherapeutic applicationsSmall molecule applicationsGeneral research applicationsNative ligandsFor full versatility: Sensor Chip CM5This sensor chip provides a high capacity to immobilize a wide range ofligands. You can employ a variety of coupling chemistries to exploit commonfunctional groups such as amino, thiol, hydroxyl, carboxyl, and aldehyde groups.For small molecules and fragments: Sensor Chip CM7Use this sensor chip when you are interested in screening and characterizingsmall molecules and fragments. It is a high capacity alternative, usefulwhen the target protein has a low concentration or is very sensitive toimmobilization conditions.To reduce background binding: Sensor Chip CM4This sensor chip has a dextran matrix similar to sensor Chip CM5 but with alower charge making it suitable for exploring alternative assay conditions.Explore alternative assay conditions: Sensor Chip CM3Shorter dextran matrix and similar charge density to Sensor Chip CM5 forexploring alternative assay conditions.If there is a need to avoid dextran: Sensor Chip C1This sensor chip has a carboxymethylated, matrix-free surface for covalentimmobilization if there is a need to avoid dextran on the surface.For antibody quantitation and characterization: Sensor Chip Protein AThe recombinant Protein A pre-immobilized on this ready-to-use sensor chipbinds antibodies, predominantly IgG from humans and mice, in the Fc regiononly. Simple and efficient surface regeneration with Glycine 1.5 (BR100354).A sensor surface for every need Our extensive range of Biacore sensor surfaces enables you to study interactionsinvolving virtually any protein and also many other biomolecules and structures,from small organic molecules up to viruses.Modified ligands, lipids or membranesFor biotinylated ligands: Sensor Chip SAThis sensor chip carries covalently attached streptavidin to give you high affinitycapture of biotinylated ligands such as proteins, peptides, nucleic acids, or carbohydrates with an orientated immobilization.For histidine-tagged molecules: Sensor Chip NTAThis sensor chip offers convenient capture of histidine-tagged molecules through metal-chelation, allowing simple and efficient surface regeneration by EDTA.For proteins in a lipid monolayer: Sensor Chip HPAThis sensor chip captures lipids as a monolayer on its hydrophobic surface.It is designed to help you easily set up a model for studying membrane-associatedproteins.For proteins in a lipid bilayer: Sensor Chip L1This lipophilic sensor chip is designed for stable capture of lipid vesicles and liposomes whilst retaining the bilayer structure of the membrane, providing a suitable system for studying transmembrane proteins.For unique surface chemistries: Sensor Chip Au and SIA Kit AuThe plain gold surface of Sensor Chip Au enables you to customize the surface for your own immobilization system. SIA Kit Au provides unmounted gold surfaces with a separate chip carrier to prepare surfaces using harsh conditions.Rapid selection of human Fab fragments: Human Fab Capture KitThis capture kit provides reagents and protocols for screening and characterizationof human Fab antibody fragments, using a Fab binder with broad specificity andstable, high capture efficiency.To capture human antibodies: Human Antibody Capture KitThis capture kit provides you with reagents and protocols for convenient captureof human or humanized IgG antibodies, providing rapid, consistent analyses withminimal assay development.For mouse IgG antibodies: Mouse Antibody Capture KitThis capture kit provides you with reagents and protocols for convenient capture ofmouse antibodies, providing rapid, consistent analyses with minimal assay development.Capture of histidine-tagged molecules: His Capture KitThis kit enables capture by an anti-histidine antibody, as a complement to captureby metal-chelation on Sensor Chip NTA.Directed capture of GST fusion proteins: GST Capture KitThis capture kit provides anti-GST antibody and reagents for the site-directedcapture of GST fusion proteins.Extended experimental range: Biotin CAPture KitBiotin CAPture Kit provides a method for reversible capture of biotinylatedmolecules and standardized regeneration. This enables work with unstable ligandsand allows analysis of different biotinylated ligands with the same sensor chip.Kits save you time and effortOur capture kits significantly reduce the time and effort you need to spend ondeveloping your assay. In addition, our coupling kits include selected reagents forcovalent attachment of your ligand.Biacore capture kits cut assay development and add consistencyThe capture approach enables orientated immobilization of ligand from a complex solution.Biacore capture kits save you time and effort by eliminating most of the assay developmentwork. They also provide consistent capture levels which are important,for example, when studying panels of antibodies.Our range gives you a number of options for capturing the most common antibodies and tags.All kits contain validated, high-quality reagents and optimized protocols.Coupling kits for a multitude of moleculesWhen your ligand is covalently attached to the sensor surface, regeneration does not removethe ligand. This can help to reduce the consumption of precious ligands. Also, covalent immobilization normally results in very stable attachment of the ligand to the surface.Coupling via primary amine groups: Amine Coupling KitAmine coupling chemistry is the most widely-applicable approach for attachingbiomolecules covalently to the surface.Defined orientation through thiol/disulfide exchange: Thiol Coupling KitYou can refine the orientation of your protein by using thiol groups instead of aminegroups, and you may even achieve more efficient coupling. Disulfide groups can alsobe introduced, which is useful for acidic proteins. This kit contains all the reagentsyou need for coupling.Immobilization buffersWith convenience in mind right from the start, GE Healthcare provides a range of immobilization buffers for the most common ligand types and immobilization conditions.Sample preparationComponents in complex sample matrices such as plasma, serum, or cell lysates may bind non-specifically to the dextran surface of sensor chips, complicating the analysis of specific binding interactions. You can minimize these effects by using NSB Reducer, which is simply added to the sample before injection.Running buffersThe recommended running buffer for your assay depends on the type of molecules used in the interaction, which assay will be run, and the type of sensor chip used. Our range of running buffers provides you with both convenience and quality, supplied in ready-to-use or concentrated form.Regeneration solutionsRegeneration is the step where bound analyte is removed from the sensor chip after analysis, without affecting the activity of the immobilized ligand. In many systems, conditions that remove analyte tend to reduce ligand activity, and finding the optimal conditions is an essential part of assay development.GE Healthcare has developed a series of regeneration solutions that meets the majority of needs. We have also simplified your search for suitable regeneration solutions by providing a Regeneration Scouting Kit, which includes small volumes of a range of regeneration solutions together with instructions giving clear guidance in the scouting process.Buffers and solutions for convenienceBuffers and solutions developed and verified to work in Biacore systems get you up and running fastExpand your expertise today Visit our Biacore training portal and get the most out of your Biacore systemwith our comprehensive applications support and training toolsSelf-training toolsGE Healthcare offers a wealth of self-training tools on the use of Biacore systems,designed to help you make the most of your investment.Getting started: Guide new users through basic hands-on training.E-learning courses: Learn online at your convenience, from the basics to advancedcalculations for measuring kinetics and affinity.Educational lecture package: Support materials for your own training course on yourpremises (For bona fide teaching purposes only).Classroom coursesTake practical and theoretical courses with personal support and comprehensivecourse literature. Extensive access to teaching resources for the hands-on sessionsand software exercises allows individual pacing with optimum support.Visit /bctraining and download all the Biacore trainingmaterial you need.Application support toolsExplore, download, and save our Tech Tips, methods, interactive trainingtutorials, calculator tools, and software to help you increase theeffectiveness of your Biacore system.Choose from our range of LabGuides andhandbooks for all the theory, workflows,protocols, recipes, and troubleshootingguides you need to help make yourexperiments a success.Visit /bcappsupportand get instant access to all our applicationsupport tools whenever you need it.Ordering Information Series S Sensor Chip CM5 Series S Sensor Chip CM7 Series S Sensor Chip CM4 Series S Sensor Chip CM3Series S Sensor Chip C1 Series S Sensor Chip NTA Series S sensor chips for Biacore 4000, Biacore A100, Biacore S200, Biacore T200, Biacore T100 and Biacore S51Series S Sensor Chip SA Series S Sensor Chip L1 Series S Sensor Chip HPA Series S Sensor Chip Protein A Sensor chipsSensor chips for other Biacore systems Sensor Chip CM5Sensor Chip CM7Sensor Chip CM4Sensor Chip CM3Sensor Chip Protein ASensor Chip NTASensor Chip L1Sensor Chip HPASensor Chip AuReagents, buffers and solutions Immobilization reagents* The use of these products in Biacore systems requires Amine Coupling Kit with Sensor Chip CM5, CM4, CM3 or C1.Capture reagentsHuman Fab Capture Kit* The use of these products in Biacore systems requires Amine Coupling Kit with Sensor Chip CM5, CM4, CM3 or C1.Reagents, buffers and solutions Regeneration solutionsRunning buffersReagents, buffers and solutions AdditivesNSB ReducerMaintenance kitsAccessories VialsCapsAccessories and compatibilitiesSample and reagent racksAdditional informationBiacore 4000 and Biacore A100 — racks and caps are not required.Biacore X, Biacore J and BIAlite™ — use any of the vials and caps listed.Miscellaneous/biacoreGE, GE monogram, BIAlite, and Biacore are trademarks of General electric Company.TWEEN is a trademark of Uniqema, a business unit of ICI Americas Inc. Kraton is a trademark of Kraton Polymers U.S. LLC. All other third party trademarks are the property of their respective owners.© 2011–2015 General Electric Company. First published Feb. 2011.GE Healthcare Bio-Sciences AB, Björkgatan 30, 751 84 Uppsala, Sweden28992438 AE 08/2015。