HisTag_融合蛋白纯化(默克新版)

His标签融合蛋白纯化常见问题解析（博进生物）1. 纯化原理His标签融合蛋白的纯化是借助于层析介质上的过渡金属离子（如Cu2+、Zn2+、Ni2+、Co2+等）与His融合蛋白上的His标签的配位作用实现目标蛋白的分离。

组氨酸（His）的残基上带有1个咪唑基团，可以和Ni2+等过渡金属离子形成配位键而选择性的结合在金属离子上，这些金属离子通过螯合配体固定在层析介质上，因此带有His标签的蛋白可以特异性的吸附在螯合了Ni2+等过渡金属离子的层析介质上，而其他不含His标签的杂质蛋白则不能吸附或仅微弱吸附在介质上。

通过提高缓冲液中的咪唑浓度进行竞争性洗脱，可以将His标签融合蛋白从层析介质上解吸附下来，从而得到较高纯度的目标蛋白。

2. His标签蛋白纯化策略His标签蛋白纯化的所有优化策略都是基于改变标签蛋白和过渡金属离子之间配位作用力的强弱，而影响配位作用力强弱的因素如下：2.1 标签长度及暴露程度最常用的His标签是6个重复的组氨酸，但在实际操作过程中，可控制在4-10个组氨酸的标签长度。

标签越长，蛋白与过渡金属离子的结合力越强。

过渡金属离子只能与蛋白表面的组氨酸集合，所以His标签暴露的程度越高，结合能力越强。

2.2 过渡金属离子半径常用于螯合His标签蛋白的过渡金属离子有Cu2+、Zn2+、Ni2+、Co2+等，金属离子的半径越小，与His标签蛋白的结合能力就越强。

上述金属离子与His标签蛋白结合能力的强弱顺序为Cu2+ >Zn2+ >Ni2+ >Co2+2.3 缓冲液条件His标签融合蛋白与过渡金属离子的螯合作用受缓冲液的pH值影响。

在中性或弱碱性（pH 7-8）环境下的螯合作用力要强于酸性环境下的作用力。

His标签融合蛋白与过渡金属离子的螯合作用还受缓冲液种类的影响。

通常磷酸盐缓冲液中的螯合作用力强于Tris-HCl缓冲液中的作用力。

由于咪唑可以和His标签蛋白竞争过渡金属离子，所以缓冲液中咪唑浓度的升高可减弱上述螯合作用力。

一：在自然条件下从大肠杆菌纯化带组氨酸的蛋白质1.推荐培养的大肠杆菌的体积：这是根据表达系统和组氨酸标记的蛋白质的性质来决定的。

Midi型号的养25-150mL的大肠杆菌。

2.收集菌的细胞：注：菌体不用的时候需要保存在-800C。

a.将过夜培养的细菌离心（6000g，40C，15min），然后小心的除掉上清。

b.制备1X LEW Buffer 和1X Elution Buffer。

3.悬浮细胞体：这步在冰上进行用1X LEW Buffer将上步离心过后的菌体吹悬，一般来说每150mL菌液加3mL的1X LEW Buffer（每湿重1g的菌体加5mL的1X LEW Buffer）。

如果菌体冻住，则需要在冰上用1X LEW Buffer将其溶解,溶解后转入15 ml的离心管中，便于进行超声裂解。

4.使裂解物澄清a.将溶菌酶添加到上面得到的溶液中（1mL的LEW混合液+20uL的50mg/mL的溶菌酶）。

然后将它放置在冰上30mins，可能的话中间偶尔混合一下。

b.超声裂解：工作2s，间歇2s，如此循环180次，总共18mins。

注：超声在冰浴上进行。

如果得到的溶液有粘着，可加入DNase1。

c.离心（13000rmp，40C，15mins），收集上清。

注：如果上清不是澄清的，进行第二次离心，是为了防止没有溶解的菌体堵塞柱子。

5.平衡PrepEase® Ni-NTA Columna.将柱子放在合适的离心管中，最好是15mL的离心管。

可以将15ml离心管插到冰上。

将柱子放到带圆孔的纸片中，放入15ml离心管中b.用2mL的1X LEW Buffer平衡柱子。

允许柱子在重力的作用下排干。

6.将样品结合到PrepEase® Ni-NTA Column将步骤4c得到的上清加到柱子里，并允许柱子在重力的作用下排干。

7.洗柱子:用1X LEW Buffer洗柱子，洗两次，每次用2mL的1X LEW Buffer。

His标签蛋白纯化实验通过实验，学习和了解 His-Tag 融合蛋白的表达、纯化原理和方法，掌握相关仪器设备的操作使用。

一、实验原理金属螯合离子亲和色谱（IMAC）是常见的亲和纯化方案之一，主要利用介质配体螯合的金属离子吸附纯化表面带组氨酸、色氨酸或半胱氨酸残基的蛋白。

His-tag 融合蛋白是目前最常见的表达方式，而且很成熟，它的优点是表达方便而且基本不影响蛋白的活性。

Ni IDA Beads 可以用于各种表达来源（如大肠杆菌、酵母、昆虫细胞和哺乳动物细胞）的组氨酸标签（6xHis-tagged）蛋白的纯化。

它是以 4%琼脂糖凝胶为基质，通过化学方法偶联了三配位的亚氨基二乙酸（IDA），螯合镍离子（Ni ）后，可以形成比较稳定的平面四边形结构，从而有更多的位点与组氨酸标签上的咪唑环继续配位，达到结合目的蛋白的效果（产品化学结构见图 1.1 所示）。

二、实验准备试剂菌种、LB 培养基、氨卞青霉素、异丙基硫代半乳糖苷(IPTG)、His-Tag 蛋白纯化试剂盒实验仪器和设备移液器、恒温振荡箱、超声破碎仪、离心机、紫外检测仪、冰箱三、实验步骤第一步、菌体制备1、取 2 支 4ml LB 培养基试管，超净台中操作。

每管加入 4ul 菌种，4ul 氨卞青霉素。

放入 37 度恒温振荡箱 180rpm，过夜培养，16 小时。

2、将菌种加入 800ml LB 培养基，800ul 氨卞青霉素，放入 37 度恒温振荡箱 180rpm培养 4 小时，OD600 约 0.6-0.8。

3、加入 IPTG 800ul，放入 37 度恒温振荡箱 180rpm 培养 4 小时。

4、离心 8000rpm 离心 10min 收集菌体，-80 度保存菌体。

第二步、菌体破碎1、将菌体取出，加入 50ml Lysis Buffer 磁力搅拌悬浮，待无明显块状即可。

2、超声 4s 停 6s，36°保护温度，超声 30min。

3、将破碎好的裂解液离心取上清（11000rpm ,20min ,4℃），准备上样。

Code No. Product No. supplied 17-5247-01 HisTrap HP 1 ml 5 × 1 ml 17-5247-05 HisTrap HP 1 ml 100 × 1 ml* 17-5248-01 HisTrap HP 5 ml 1 × 5 ml 17-5248-02 HisTrap HP 5 ml 5 × 5 ml 17-5248-05 HisTrap HP 5 ml 100 × 5 ml* * Pack size available by special order.Connector kitConnectors 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.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. Further Information 1711. Ordering Information 18p. 21. DescriptionMedium propertiesHisTrap HP 1 ml and 5 ml columns are prepacked with Ni Sepharose High Performance, which consists of 34 µm highly cross-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 are exposed on the proteinsurface. Additional histidines, such as in the case of (histidine)6 -tag,increase affinity for Ni2+ and generally make the histidine-tagged proteinthe strongest binder among other proteins in e.g. an E. coli extract. Column propertiesHisTrap HP columns are made of biocompatible polypropylene that doesnot interact with biomolecules. Columns are delivered with a stopper on the inlet and a snap-off end on the outlet. The columns have porous top and bottom frits that allow high flow rates. They cannot be opened or refilled. Columns can be operated with either a syringe and the supplied luer adapter, a peristaltic pump, or a chromatography system such asÄKTAdesign or FPLC System.Note: To prevent leakage, ensure that the adapter is tight.p. 3Table 1. HisTrap HP characteristics.Matrix Highly cross-linked spherical agarose, 6% Average particle size 34 µmMetal ion capacity ~15 µmol Ni2+/ml mediumDynamic bindingcapacity* At least 40 mg (histidine)6-tagged protein/ml mediumColumn volumes 1 ml or 5 mlColumn 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 respectively Max. flow rates 4 and 20 ml/min for 1 and 5 ml column respectively Max back pressure††0.3 MPa, 3 barCompatibility during use Stable in all commonly used buffers, reducing agents,denaturants, and detergents. See Table 2.Chemical stability§§§0.01 M HCl, 0.1 M NaOH. Tested for 1 week at 40 ºC.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§§§short term (< 2 hours): 2–14long term (< 1 week): 3–12Storage 20% ethanolStorage temperature +4 to +30 ºC* Dynamic binding capacity conditions:Sample: 1 mg/ml (histidine)6-tagged pure protein (Mr28 000 or 43 000) inbinding buffer (QB, 10% determination) or (histidine)6-tagged proteinbound from E. coli extractColumn volume: 0.25 ml or 1 mlFlow rate: 0.25 ml/min or 1 ml/minBinding 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 7.4 Note: Dynamic binding capacity is protein-dependent.††H2O at room temperature§§§Ni2+-stripped mediumThe Ni2+-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).p. 4Table 2. Ni Sepharose High Performance is compatible with the following compounds, at least at the concentrations given.Reducing agents* 5 mM DTE5 mM DTT20 mM ß-mercaptoethanol5 mM TCEP10 mM reduced glutathioneDenaturing agents 8 M urea††6 M guanidine hydrochloride††Detergents 2% Triton X-100 (nonionic)2% Tween 20 (nonionic)2% NP-40 (nonionic)2% cholate (anionic)1% CHAPS (zwitterionic)Other additives 500 mM imidazole20% ethanol50% glycerol100 mM Na2SO41.5 M NaCl1 mM EDTA§§§60 mM citrate§§§Buffer substances 50 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 4††* See Notes and blank run, p. 10–11.††Tested for 1 week at +40 °C.§§§The 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 smallexcess of MgCl2 before centrifugation/filtration of the sample. Note that stripping effectsmay vary with applied sample volume.p. 52. General considerationsHisTrap HP is supplied precharged with Ni2+ ions. In general, Ni2+ is thepreferred metal ion for purification of recombinant histidine-taggedproteins. Note, however, that in some cases it may be wise to test othermetal ions, e.g. Zn2+ and Co2+, as the strength of binding depends onthe nature of the histidine-tagged protein as well as the metal ion (seeOptimization).We recommend binding at neutral to slightly alkaline pH (pH 7–8) in thepresence 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 Table 2.Including salt e.g. 0.5–1.0 M NaCl in the buffers and samples, eliminatesion-exchange effects but can also have a marginal 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 the sample (generally, at the same concentration as in the wash buffer). At somewhat higher concentrations, imidazole may also decrease the binding of histidine-tagged proteins. The imidazole concentration must therefore be optimized to ensure the best balance of high purity (low binding of host cell proteins) and high yield(binding of histidine-tagged target protein). This optimal concentration is different for different histidine-tagged proteins, and is usually slightly higher for Ni Sepharose High Performance than for similar IMAC media on themarket (see Data File 18-1174-40). Use highly pure imidazole; such imidazole gives essentially no absorbance at 280 nm.As alternatives to imidazole elution, histidine-tagged proteins can be eluted from the medium by several other methods or combinations of methods – a lowering of pH within the range of 2.5–7.5 can be used, for example. At pH values below 4, metal ions will be stripped off the medium.Note:If the proteins are sensitive to low pH, we recommend collection of the eluted fractions in tubes containing 1 M Tris-HCl, pH 9.0(60–200 µl/ml fraction) to restore the pH to neutral.p. 6Chelating agents such as EGTA or EDTA will also strip metal ions from 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 ona HiTrap™ Desalting, a PD-10 Desalting Column, or HiPrep™ 26/10 Desalting, (see Table 4).Leakage of Ni2+ from Ni Sepharose High Performance is very low underall normal conditions, lower than for other IMAC media tested. For applications where extremely low leakage during purification is critical, leakage can be even further reduced by performing a blank run (see page 11).Likewise, a blank run should also be performed before applying buffers/ samples containing reducing agents (see page 11).Whatever conditions are chosen, HisTrap HP columns can be operated witha syringe, peristaltic pump, or chromatography system.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.3. OperationBuffer 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 before use.Use high purity imidazole as this will give very low or no absorbance at 280 nm.If the recombinant histidine-tagged protein is expressed as inclusion bodies, include 6 M Gua-HCl or 8 M urea in all buffers and sample. On-column refolding of the denatured protein may be possible.p. 7Recommended conditionsBinding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 20–40 mMimidazole, pH 7.4 (The optimal imidazoleconcentration is protein-dependent; 20–40 mM issuitable for many proteins.)Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 500 mMimidazole, pH 7.4 (The imidazole concentrationrequired for elution is protein-dependent). Sample 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 bufferby: Adding buffer, NaCl, imidazole, and additives from concentratedstock solutions; by diluting the sample with binding buffer; or by bufferexchange, (see Table 4). 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 isessential to include imidazole at a low concentration in the sample andbinding buffer (see Optimization).p. 8p. 9T a b l e 4. P r e p a c k e d c o l u m n s f o r d e s a l t i n g a n d b u f f e r e x c h a n g e .C o d e N o C o l u m nL o a d i n g v o l u m e E l u t i o n v o l u m e C o m m e n t sA p p l i c a t i o n17-1408-01 H i T r a p 0.1–1.5 m l 1.3–4.0 m l P r e p a c k e d w i t h F o r d e s a l t i n g a n d b u f f e r D e s a l t i n g S e p h a d e x ™ G -25 e x c h a n g e o f p r o t e i n S u p e r f i n e . R e q u i r e s e x t r a c t s (M r >5000). a s y r i n g e o r p u m p t o r u n . 17-5087-01 H i P r e p U p t o 15–20 m l P r e p a c k e d w i t h F o r d e s a l t i n g a n d 26/10 15 m l S e p h a d e x G -25 b u f f e r e x c h a n g e o f D e s a l t i n g F i n e . R e q u i r e s a p r o t e i n e x t r a c t s p u m p t o r u n . (M r >5000).17-0851-01 P D -10 2.5 m l 3.5 m l P r e p a c k e d w i t h F o r d e s a l t i n g a n d D e s a l t i n g S e p h a d e x G -25. b u f f e r e x c h a n g e R e q u i r e s o n l y o f p r o t e i n e x t r a c t s g r a v i t y t o r u n . (M r >5000).17-0855-01 N I C K ™ 0.1 m l 0.4 m l P r e p a c k e d w i t h F o r s e p a r a t i o n o f S e p h a d e x G -25. p r o t e i n s (M r >5000) a n d n i c k t r a n s l a t e d D N A f r o m r a d i o l a b e l l e d n u c l e o t i d e s n o t s h o r t e r t h a n 120 m e r s , a n d s i m i l a r s e p a r a t i o n s .17-0853-01 N A P ™-5 0.5 m l 1.0 m l P r e p a c k e d w i t h F o r p u r i f i c a t i o n o f 17-0854-01 N A P -10 1.0 m l 1.5 m l S e p h a d e x G -25 p r o t e i n s (M r >5000),17-0852-01 N A P -25 2.5 m l 3.5 m l D N A g r a d e . D N A a n d o l i g o - R e q u i r e s o n l y n u c l e o t i d e s g r e a t e rg r a v i t y t o r u n . t h a n 10 b a s e s i n l e n g t h .Purification1. Fill the syringe or pump tubing with distilled water. Remove the stopperand connect the column to the syringe (use the adapter provided),laboratory pump or chromatography system tubing “drop-to-drop” toavoid introducing air into the system.2. Remove the snap-off end at the column outlet.3. Wash the column with 3–5 column volumes of distilled water.4. Equilibrate the column with at least 5 column volumes of binding buffer.Recommended flow rates are 1 ml/min or 5 ml/min for the 1 and 5 mlcolumns respectively.In some cases a blank run is recommended before final equilibration/sample application (see page 11).5. Apply the pretreated sample using a syringe or a pump.6. Wash with binding buffer until the absorbance reaches a steadybaseline (generally, at least 10–15 column volumes).Note:Purification results are improved by using imidazole in sample and binding buffer (see Optimization).7. Elute with elution buffer using a one-step or linear gradient.Five column volumes are usually sufficient if the protein of interest iseluted by a one-step gradient. A shallow gradient, e.g. a linear gradient over 20 column volumes or more, may separate proteins with similarbinding strengths.Note:If imidazole needs to be removed from the protein, use HiTrap Desalting, a PD-10 Desalting Column, or HiPrep 26/10 Desaltingdepending on the sample volume (see Table 4).Note:Ni Sepharose High Performance is compatible with reducing agents.However, removal of any weakly bound Ni2+ ions by performing ablank run without reducing agents (as described on page 11) beforeapplying buffer/sample including reducing agents is recommended.Do not leave HisTrap HP columns with buffers including reducingagents when not in use.p. 10Note:Leakage of Ni2+ from Ni Sepharose High Performance is low under all normal conditions. The leakage is lower than for other IMAC mediatested (see Data File Ni Sepharose High Performance, 18-1174-40).For very critical applications, leakage during purification can be even further diminished by performing a blank run (as described below)before loading sample.Blank run:Use binding buffer and elution buffer without reducing agents.1. Wash the column with 5 column volumes of distilled water (to removethe 20% ethanol).2. Wash with 5 column volumes of elution buffer.3. Equilibrate with 10 column volumes of binding buffer.4. OptimizationImidazole 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 the sample (generally, at the same concentration as in the wash buffer). At somewhat higher concentrations, imidazole may also decrease the binding of histidine-tagged proteins. The imidazole concentration must therefore be optimized to ensure the best balance of high purity (low binding of host cell proteins), and high yield (binding of histidine-tagged target protein). This optimal concentration 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 Ni Sepharose High Performance, 18-1174-40). Finding the optimal imidazole concentration for a specific histidine-tagged protein is a trial-and-error effort, but 20–40 mM in the binding and wash buffers is a good starting point for many proteins. Use a high purity imidazole, such imidazole gives essentially no absorbance at 280 nm.Ni2+ is usually the first choice metal ion for purifying most (histidine)6-taggedrecombinant proteins from nontagged host cell proteins, and also the ion most generally used. Nevertheless, it is not always possible to predict whichp. 11metal ion will be best for a given protein. The strength of binding between a protein and a metal 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 optimize separation conditions is to use HiTrap IMAC HP 1 ml columns, which are packed with IMAC Sepharose High Performance (not charged with metal ions). Each column can be charged with different metal ions, e.g. Cu2+, Co2+, Zn2+, Ca2+, or Fe2+. Instructions are included with each column.A study to compare the purification of six (histidine)6-tagged recombinantproteins, including three variants of maltose-binding protein, with different metal ions has indicated that Ni2+ generally gives best selectivity between (histidine)-tagged and nontagged host-cell proteins (see Application Note 18-1145-18).5. Stripping and rechargingNote:The column does not have to be stripped and recharged between each purification if the same protein is going to be purified; it issufficient to strip and recharge it after 5–7 purifications, dependingon 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 of binding 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 of 0.1 MNiSO4 in distilled water on HisTrap HP 1 ml and5 ml column, respectively.Salts of other metals, chlorides, or sulfates, may also be used (seeOptimization). Wash with 5 column volumes distilled water, and 5 column volumes binding buffer (to adjust pH) before storage in 20% ethanol.p. 126. Cleaning-in-placeWhen an increase in back-pressure is seen, the column can be cleaned. Before cleaning, strip off Ni2+ ions using the recommended 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 following methods;• Remove ionically bound proteins by washing the column with several column volumes of 1.5 M NaCl; then wash the column with approx. 10column volumes of distilled water.• Remove precipitated proteins, hydrophobically bound proteins, and lipoproteins by washing the column with 1 M NaOH, contact time usually 1–2 hours (12 hours or more for endotoxin removal). Then wash thecolumn with approx. 10 column volumes of binding buffer, followed by5–10 column volumes of distilled water.• Remove hydrophobically bound proteins, lipoproteins, and lipids by washing the column with 5–10 column volumes 30% isopropanol forabout 15–20 minutes. Then wash the column with approx. 10 columnvolumes of distilled water.Alternatively, wash the column with 2 column volumes of detergent in a basic or acidic solution. Use, for example, 0.1–0.5% nonionic detergentin 0.1 M acetic acid, contact time 1–2 hours. After treatment, alwaysremove residual detergent by washing with at least 5 column volumes of 70% ethanol. Then wash the column with approx. 10 column volumes of distilled water.7. Scaling-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 in HisTrap HP columns, is supplied preswollen in 25 and 100 ml lab packs (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.p. 138. StorageStore HisTrap HP columns in 20% ethanol at +4 to +30 °C.9. TroubleshootingThe following tips may be of assistance. If you have any further questions about your HisTrap HP column, please visit/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 afterelution) 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-exchanged toa 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 a 0.22 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 addDNase I to 5 µg/ml, Mg2+ to 1 mM, and incubate on ice for 10–15 min.Alternatively, draw the lysate through a syringe needle several times. p. 14Protein 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 mMß-mercaptoethanol, 1–3 mM DTT or DTE (up to 5 mM is possible butdepends on the sample and the sample volume), 5 mM TCEP, 10 mMreduced glutathione, 8 M urea, or 6 M Gua-HCl. Mix gently for 30 min toaid solubilization of the tagged protein (inclusion bodies may requirelonger mixing). Note that Triton X-100 and NP-40 (but not Tween) have a high absorbance at 280 nm. Furthermore, detergents cannot be easilyremoved 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 todetermine the optimal elution conditions.• Protein has precipitated in the column: For the next experiment, decrease amount of sample, or decrease protein concentration byeluting with linear imidazole gradient instead of imidazole steps. Trydetergents or changed NaCl concentration, 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 theNaCl concentration.• Concentration of imidazole in the sample and/or binding buffer is too high: The protein is found in the flow-through material. Decrease theimidazole 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 strong reducing agents are not presentin the sample at too high concentration, and that the concentration ofimidazole is not too high.p. 15SDS-PAGE of samples collected during the preparation of the bacterial 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 by measuring the release ofnucleic acids at A260. Addition of lysozyme (up to 0.1 volume of a 10 mg/mllysozyme solution in 25 mM Tris-HCl, pH 8.0) prior to sonication mayimprove results. Avoid frothing and overheating as this may denaturethe target protein. Over-sonication can also lead to copurification 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 usingcommon denaturants such as 4–6 M Gua-HCl, 4–8 M urea, or strongdetergents.Prepare buffers containing 20 mM sodium phosphate, 8 M urea,or 6 M Gua-HCl, and suitable imidazole concentrations, pH 7.4–7.6.Buffers with urea should also include 500 mM NaCl. Use these buffersfor sample preparation, as binding buffer and as elution buffer. Forsample preparation and binding buffer, use 10–20 mM imidazole or theconcentration selected during optimization trials (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 Table 2).• Contaminants have high affinity for nickel ions: Elute with a stepwise or linear imidazole gradient to determine optimal imidazoleconcentrations to use for binding and for wash; add imidazole to thesample in the same concentration as the binding buffer. Wash beforeelution with binding buffer containing as high concentration of imidazole as possible, without causing elution of the tagged protein. A shallowimidazole gradient (20 column volumes or more), may separate proteins with similar binding strengths. If optimized conditions do not removecontaminants, further purification by ion exchange chromotographyp. 16(HiTrap Q HP or HiTrap 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 or 2% Tween 20), or add glycerol (up to 50%)to the wash buffer to 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 that chelating or strong reducingagents are not present in the sample at a too high concentration, andthat the concentration 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 HP 1 ml columns together or change to a HisTrap HP 5 ml column.10. Further informationVisit /hitrap for further information. Several handbooks also contain useful information, see ordering information.p. 1711. Ordering InformationProduct No. supplied Code No.HisTrap HP 5 × 1 ml 17-5247-01 HisTrap HP 100 × 1 ml * 17-5247-05 HisTrap HP 1 × 5 ml 17-5248-01 HisTrap HP 5 × 5 ml 17-5248-02 HisTrap HP 100 × 5 ml * 17-5248-05 Related products No. supplied Code No.Ni Sepharose High Performance 25 ml 17-5268-01 Ni Sepharose High Performance 100 ml 17-5268-02 HiTrap Desalting 5 × 5 ml 17-1408-01 HiTrap Desalting 100 × 5 ml * 11-0003-29 PD-10 Desalting Column 30 17-0851-01 HiPrep 26/10 Desalting 1 × 53 ml 17-5087-01 HiPrep 26/10 Desalting 4 × 53 ml 17-5087-02 HisTrap FF 5 × 1 ml 17-5319-01 HisTrap FF 100 × 1 ml * 17-5319-02 HisTrap FF 5 × 5 ml 17-5255-01 HisTrap FF 100 × 5 ml * 17-5255-02 HisTrap FF crude 5 × 1 ml 11-0004-58 HisTrap FF crude 100 × 1 ml * 11-0004-59 HisTrap FF crude 5 × 5 ml 17-5286-01 HisTrap FF crude 100 × 5 ml * 17-5286-02 HisTrap FF crude Kit 1 kit 28-4014-77 HisPrep™ FF 16/10 1 × 20 ml 17-5256-01 * Pack size available by special order.p. 18。

His标签融合蛋白纯化步骤（Ni-NTA琼脂糖凝胶亲和层析纯化法）1. 缓冲液配制◆L ysis Buffer 1 (under native condition)0.5mM Tris.HCl0.5M NaCl5%(w/v) glycerol10mM Imidazol100mg(1mg/ml)lysozyme (Purification of 6xHis-tagged proteinsfrom E.coli)1% Nonidet P40 (NP40 = Igepal CA-630 )0.25% Tween 20 （or Triton 100）0.02% NaN3 (Optional)2 tablets of protease inhibitor cocktail (EDTA free，Recommended)200 µg(2µg/ml) RNase A (Optional)1mg (10µg/ml) DNase1 (Optional)50 mM NaF (Optional)1mM Na3VO4 (Optional)+ ddH2O to 100ml, Adjust pH to 8.0 using NaOH此lysis buffer 适用于从E.coli、哺乳动物细胞和昆虫细胞中纯化带His标签的蛋白质。

仅在用于裂解E.coli细菌时，加入溶菌酶。

Na3VO4是磷酸酶抑制剂，保护磷酸化的蛋白不被磷酸酶还原。

NaF是酯酶抑制剂，保护脂蛋白不被酯酶降解。

注意：当使用镍琼脂糖凝胶纯化带His标签的蛋白时，缓冲液中不能加EDTA，因EDTA能使镍从螯合物上脱离，从而使分离介质失去效果。

◆L ysis Buffer 2(under denature condition)50 mM Tris-Cl8 M Urea (or 6 M Gu-HCl)10mM Imidazole0.05% Tween 20Adjust pH to 8.0 using NaOH◆D ialysis/Tev cleavage Buffer50 mM Tris-Cl,pH8.0100 mM NaCl (depends on solubility of protein)2.5% glycerol0.5mM EDTA0.5mM DTT or TCEP◆缓冲液A：50mM Tris.HCl0.5M NaCl5% glycerol0.05% Tween 20用高浓度HCl调节pH值至8.0。

组氨酸(His)标签蛋白的纯化His-Tag融合蛋白是目前最常见的表达方式，而且很成熟，它的优点是表达方便而且基本不影响蛋白的活性，无论是表达的蛋白是可溶性的或者包涵体都可以用固定金属离子亲和色谱（IMAC）纯化。

IMAC(Immobilized Metal-ion affinity chromatography)是Porath et al.1975年用固定IDA作为配基的填料螯合过渡金属铜、镍、钴或锌离子，可以吸附纯化表面带组氨酸、色氨酸或半胱氨酸残基的蛋白，1987年Smith et al. 发现带有几个组氨酸或色氨酸小肽和螯合金属离子的IDA-sephadex G-25作用力更强，此前在1986年他和他的合作者用Ni2+-IDA-sephadex G-25亲和纯化在氨基端带组氨酸和色氨酸的胰岛素原。

同年1987年Hochuli et al.发现带有相连组氨酸的多肽和Ni2+-NTA填料作用力更强于普通的肽，1988年他第一次用这样的方法纯化了带六个组氨酸标签的多肽，无论是在天然还是变性条件下一次亲和纯化都得到很好效果，此后表达带六个组氨酸标签的蛋白配合IMAC变得非常普遍，相对而言，不带标签的蛋白纯化就非常困难，所以表达带六个组氨酸标签的蛋白配合IMAC纯化变成最常用而且最有效的研究蛋白结构和功能的有力手段。

1986年Porath et al.还发现Fe3+-IDA-sephadex G-25可以用于磷酸化蛋白的纯化，而后发现Ga3+-IDA也有同样的效果，这样螯合这两种金属离子的填料就有效用于磷酸化多肽的富集和纯化，同时IMAC也可以用于纯化各种和金属离子结合的多肽，应用非常广泛。

Ni柱中的氯化镍可以与有HIs（组蛋白）标签的蛋白结合，也可以与咪唑结合。

步骤是：过柱子前可以选择Ni柱重生，也就是往柱子里倒氯化镍，一个柱长体积就行了，然后平衡柱子，拿你自己的buffer，给蛋白提供最适的环境，我一般平衡4个柱长，然后蛋白上样，你可以让他自己挂，这样挂柱子的效果好一些，如果流速太慢，可以加个恒流泵，但是一定不能太快，太快挂柱效果差，当然你也可以选择循环挂柱，就是恒流泵的一头接你装蛋白的烧杯，从柱子中留下来的液体还用同一个烧杯接回去。

His融合蛋白纯化中常见问题解答集锦His-tag是专门设计用于重组蛋白质的纯化，与其他标签相比有很多明显优势，是目前用于纯化的融合标签中使用最为广泛的一种。

Tag虽然简单，但是做过的人都知道，融合蛋白的纯化并非简单。

纯化介质有10多种，应该如何选择？洗脱的标签蛋白杂带较多是什么原因？如何优化？甚至洗脱产物中没有目标蛋白原因为何？策略如何？GE蛋白质纯化专家就His-Tag融合蛋白在纯化过程中常常遇到的问题进行解答，不容错过！对于HIS标签蛋白纯化的产品，如何选择？我公司为纯化组氨酸标记的蛋白质提供了全面的解决方案，产品种类丰富应用广泛(见图1)，下面一一介绍其特点和应用。

图1 His产品的选择Ni Sepharose High Performance用于高分辨率纯化的预带电荷的介质Ni Sepharose High Performance，通过耦联Ni使介质预带电荷，使用非常方便。

和其它厂家的同类产品比较，它具有高载量和低的Ni脱落的特点，低的镍脱落保证的HIS-标记的蛋白质在多次重复的纯化中保持可靠的载量。

同时Ni Sepharose High Performance填料的颗粒大小只有34um，具备最高的分辨率和最低的样品稀释。

Ni Sepharose High Performance除了有散装填料形式，也具有HISTrap1Ml和5Ml的实验室预装柱,可以连接AKTA仪器，蠕动本以及手动注射器操作,使用非常方便。

His MultiTrap HP 96孔过滤板用于高通量筛选和多样品的平行纯化，预装了Ni Sepharose High Performance的填料，未澄清的细胞裂解液可以直接上样，具有高重复性，操作简单方便快捷的特点。

His SpinTrap柱用于少量的蛋白质制备，预装了100ul的HP填料，可以直接上澄清和未澄清的样品，可用于细菌细胞裂解液的筛选和优化纯化条件，与标准离心机使用，一次纯化仅需10分钟。

变性条件下从大肠杆菌中纯化多聚组氨酸标签蛋白（主要以包涵体的形式表达）的样品制备1、用1× PBS重悬细胞沉淀（约每毫升沉淀加5ml 1× PBS），并按上述方法进行超声破菌。

2、12000 rpm离心10 min收集包涵体。

若有必要，用1 × PBS洗包涵体几次。

3、用Binding/Wash Buffer（约每毫升沉淀加5ml 1× PBS）溶解包涵体，并在室温下孵育30～60分钟。

若使沉淀充分溶解，有必要进行机械或超声均质。

4、12000rpm离心30min，取上清至一干净管中。

His标签蛋白的重力纯化流程1ml柱子的总体积为10ml，只需加入介质。

如果样品体积大于柱子体积，可重复利用，注意不要超过树脂的结合能力。

1、平衡柱子的工作温度。

应在室温或4℃下进行纯化。

2、取出底帽，倒出多余的液体，直立固定好柱子，让柱子顶部朝上。

3、用2倍树脂体积的Binding/Wash Buffer平衡柱子，以0.5～1 ml/min的流速过柱。

4、从柱子上部加入经Binding/Wash Buffer处理的大肠杆菌裂解物或蛋白提取物，收集流出液。

若需要，让流出液重新过柱一次，以最大限度地提高结合力。

5、用两倍树脂体积的Binding/Wash Buffer洗涤树脂并收集流出液。

重复该步骤，用一新的收集管收集流出液。

直到流出液的吸光度在280 nm基线处。

6、用两倍树脂体积的Elution Buffer将His标签蛋白从树脂上洗脱下来。

重复此步骤两次，并单独收集每次洗脱出来的液体。

7、用Modified Coomassie Bradford Assay Kit（No SK3041）。

洗脱的蛋白可直接进行SDS-PAGE分析。

注意：洗脱获得的蛋白可用凝胶过滤（如No BSP090 gravity Desalting Column）或透析去除咪唑以便后续应用。

SDS-PAGE分析前，含6M盐酸胍的样品必须用含8 M尿素的缓冲液透析。

蛋白纯化常见问题：His标签1、常用的His标签蛋白纯化填料，Ni Sepharose 6FF和Ni Sepharose HP有什么区别，应该如何选？答：HP：High Performance，高性能，填料平均颗粒大小约34 μm，颗粒细能带来高分辨率的分离纯化。

HP 纯化后峰更窄，样品浓度高，节省了浓缩步骤的时间，但会带来更高的反压，因此流速更慢。

FF：Fast Flow，快流速，填料平均颗粒大小约90 μm，较 HP 颗粒粗，流速快，具有良好的分辨率及轻松实现规模扩大的分离纯化，同样适用于重力流下的蛋白纯化和多孔板筛选。

2、HisTrap FF Crude 和 HisTrap excel 的特点是什么？应该如何选择？答：HisTrap FF Crude 的滤膜孔径更大，裂解液无需离心过滤处理，可以直接上样，适合样品不稳定或者比较粘稠容易造成柱子堵塞的料液。

例如如果纯化包涵体蛋白较多时，可以优先考虑FF Crude （预装柱）或 FF。

HisTrap excel 最大的特点是镍脱落极低，且能耐受100 mM EDTA，特别适合真核外泌蛋白（如培养基中含有螯合剂）。

无需脱镍，即可直接使用 NaOH 清洗，防止交叉污染且有更长的使用寿命。

3、 His 标签蛋白纯化实验中，常用的缓冲液成分是什么？答：建议缓冲溶液成分如下· 结合缓冲溶液：FF、 HP系列： 20mM磷酸钠缓冲溶液， 500mM NaCl(避免非特异电荷吸附)，20-50mM 咪唑(提高浓度，可以增加纯度但会降低收率)， pH7.4 (可以根据实际情况调节pH)。

Talon 系列：20mM 磷酸钠缓冲溶液，500mM NaCl，10-20mM咪唑， pH7.4。

Excel 系列： 20mM 磷酸钠缓冲溶液， 500mM NaCl， pH7.4 (结合缓冲液中一般不添加咪唑，在wash缓冲液中可根据样品性质加入 0-30 mM 咪唑)· 洗脱缓冲溶液：20mM 磷酸钠缓冲溶液，500mM NaCl，500mM 咪唑，pH 7.44、纯化得到的组分中没有或很少目的 His 标签蛋白，怎么回事？答：若目的His 标签蛋白正常表达（使用抗His 标签的抗体进行WB 检测）且充分释放至上清中，确认蛋白是流穿还是未被洗脱：若 His 标签蛋白流穿：· 样品或结合缓冲液的条件不合适，注意螯合剂或强还原剂以及咪唑的浓度。