计算溶液所需的质量、体积或浓度。
GST标签蛋白纯化试剂盒 (pH 8.0)
有货
为什么选择此级别
BioReagent,用于蛋白分析 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
2-8°C储存,室温,禁止冷冻。低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
GST，即谷胱甘肽-S-转移酶，与目的蛋白融合表达后，被称为GST标签重组蛋白、GST标签蛋白或GST融合蛋白。GST标签重组蛋白可以通过GST特异性地结合到GST Affinity Resin，而其它蛋白则不能被结合，含GST标签的重组蛋白可被过量的还原型谷胱甘肽(GSH)溶液洗脱，从而实现GST标签蛋白的分离和纯化。由于纯化过程始终保持在温和的非变性条件下，得到的目的蛋白可以保持其自身的生物学活性，因此可以用于常规的结构和功能研究、抗体制备、以及蛋白与蛋白相互作用、蛋白与核酸相互作用等方面的研究。本试剂盒不仅可以用于大肠杆菌表达的GST标签重组蛋白的纯化，也可以用于哺乳动物细胞、昆虫细胞及杆状病毒等其它表达系统中表达的GST标签重组蛋白。GST标签重组蛋白在大肠杆菌中表达时具有可溶性高，并且容易保持目的蛋白完整活性等优点。许多真核蛋白在大肠杆菌中是以包涵体的形式表达的，但当它们在大肠杆菌中与GST融合表达时，相当一部分可以实现可溶性表达，从而有利于后续的纯化。如果在GST标签和目的蛋白之间含有位点特异性的蛋白酶识别位点，如 TEV Protease (rp155927) 等，则可用相应的蛋白酶切除GST标签。通常在目的蛋白的N端加上GST标签可以保留GST的酶活性，从而便于使用GST Affinity Resin进行GST标签重组蛋白的纯化。核心成分是一种GST标签蛋白纯化树脂，结构上以4%交联的琼脂糖凝胶为基质，通过12个原子的间隔臂，用化学方法共价结合还原型谷胱甘肽制作而成。该设计使树脂的纯化效率得到了较大提高，使其每毫升基质(4%交联琼脂糖)可承载>20 mg GST融合蛋白。具有载量高，特异性强，特别适合大规模纯化和初筛实验。GST Affinity Resin凝胶的颗粒直径为45-165 μm。可耐受的最大压力为0.1 MPa，约合1 bar。GST Affinity Resin储存在含20%乙醇的1×PBS中，10 mL总体积中5 mL为琼脂糖凝胶、5 mL为液体。使用时宜把琼脂糖凝胶充分重悬后再吸取。实际使用时的最大结合量取决于待纯化的GST标签重组蛋白的分子量大小，分子量越大则最大结合容量越大，分子量越小则最大结合容量越小。本试剂盒最多可纯化10种带有GST标签的重组蛋白，每种蛋白的纯化量约为10 mg。
产品特点
1、 快速、高效：本试剂盒使用了GST标签蛋白纯化树脂，挂柱效率高，缩短操作时间的同时可以有效的避免在长时间操作过程中目的蛋白的降解或变性，充分保证目的蛋白的活性。
2、 便捷：提供了GST标签蛋白纯化时所需的相关试剂和亲和层析柱空柱管，为GST标签蛋白的纯化带来了极大的便利。
3、 载量高：每毫升基质可承载>20 mg GST融合蛋白。
特异性强：可以一步纯化各种表达系统中谷胱甘肽-S-转移酶、谷胱甘肽依赖性蛋白和谷胱甘肽重组衍生物。
使用说明
一、 样品准备
1、 细菌或酵母表达的可溶性蛋白
(1) 挑取单菌落到含有适合抗性的培养基中，根据载体使用说明，加入相应浓度的诱导剂诱导相应的时间。
(2) 表达结束后，将培养液转移到离心瓶中，7000 rpm (7500 ×g)，离心15 min收集菌体，然后按照菌体与裂解液比例为1 : 10 (W/V) 加入裂解液，根据蛋白易降解性质可选择性加入终浓度为1 mM的PMSF，也可加入其他蛋白酶抑制剂，但不能影响目的蛋白与填料的结合 。根据需求可加入溶菌酶(如果表达的宿主细胞内含pLysS或pLysE，可以不加溶菌酶)。
(3) 将菌体沉淀悬浮起来 (如果菌液浓度高，也可考虑加入10 μg/mL RNaseA和5 μg/mL DNaseI)，混匀，放置于冰上，然后冰水混合物上超声破碎细胞，至菌液基本保持澄清。
(4) 将澄清的破碎液转移至离心管中，10000 rpm (15000 ×g)，4℃离心20-30 min。取上清，置于冰上备用，进行下一步纯化。
2、 酵母、昆虫和哺乳细胞分泌表达可溶性蛋白
将细胞培养液转移至离心瓶，5000 rpm (3800 ×g)，离心10 min，收集上清，可直接上柱使用。对于大量体积的上清，需加入硫酸铵沉淀浓缩后，蛋白还需用平衡液透析后才能加入柱子。
二、 缓冲液的准备
1、 10×GSH溶液的配制：把试剂盒提供的184 mg GSH用6 mL本试剂盒提供的洗脱缓冲液溶解并混匀，即为10×GSH溶液。配制好的10×GSH溶液-20℃保存，至少一年有效。
2、 1×GSH洗脱缓冲液的配制：按照9:1的比例混合适量洗脱缓冲液和10×GSH溶液，例如9 mL洗脱缓冲液和1 mL 10×GSH溶液混合，混合后的溶液即为1×GSH洗脱缓冲液。由于GSH在溶液中容易被氧化而失效，洗脱缓冲液宜新鲜配制，配制好的洗脱缓冲液4℃保存，两周内有效。
三、 样品纯化
样品在上样前建议离心或用0.22 μm或0.45 μm滤膜过滤，减少杂质，提高蛋白纯化效率和防止堵塞柱子。缓冲液在使用前最好用0.22 μm或者0.45 μm滤膜过滤。
A. 重力柱法纯化
1、 重力柱的装填
(1) 取层析空柱，装入下垫片，加入适量纯水润洗柱管和垫片，关闭下出口。
(2) 将GST亲和树脂充分混匀，用移液器吸取适量浆液加入重力柱中(填料实际体积占悬液的一半，如吸取1 mL悬液，其实际体积为0.5 mL，则最大载量约为10 mg)，打开下出口流干保护液。
(3) 加入适量纯水冲洗填料，待柱管中液体重力流干后，关闭下出口。
(4) 加入润洗后的上垫片，确保垫片与填料之间没有空隙，且保持水平。
(5) 装填好的重力柱可以直接加入Lysis Buffer进行平衡，暂不使用时则加入保护液，4℃保存。
2、 平衡
将装填好GST亲和树脂的重力柱用2-5 CV(CV是指沉降后的树脂体积，以下内容提到的CV不再做此注解)裂解缓冲液进行平衡，重复2-3次。
3、 上样
将样品加到平衡好的重力柱中，样品缓慢流出，保证样品和填料充分接触，收集流出液，可以反复上样增加结合效率(如果目标蛋白不稳定，建议样品置于冰上)。
4、 洗杂
使用5-10 CV的裂解缓冲液洗涤柱子，去除非特异性吸附的杂蛋白，收集洗杂液。洗柱及下一步洗脱过程中可以用Bradford法简单快速地检测每次洗涤液和洗脱液中的蛋白含量，从而考虑增加或减少洗涤和洗脱的次数。
5、 洗脱
使用5-10 CV的1×GSH洗脱缓冲液洗脱目的蛋白。分段收集，每1-5个CV收集一管，分别检测，既可以保证所有结合的目的蛋白被洗脱，又可以得到高纯度和高浓度的蛋白。
B. 孵育法纯化
1、 树脂准备
根据纯化的样品量，取适量GST 亲和层析介质加入离心管中，1000 rpm离心1 min，小心的吸弃上清(保持低速离心，以免损坏树脂)；也可加入重力柱中，流干保护液。
2、 平衡
向离心管中加入5 CV裂解液，轻柔上下翻动混匀，1000 rpm 离心1 min后，小心的去除上清；如使用重力柱，则直接在重力柱中清洗，直接重力流干洗涤液；重复两次以上。
3、 树脂结合目的蛋白
将准备好的样品加入到平衡好的树脂中，封闭离心管或重力柱管，旋转振荡孵育，室温30 min-2 h或2-8℃ 2-4 h，可适当延长时间，使混合液与树脂充分混匀(如果目标蛋白不稳定，建议置于2-8℃环境中结合)。1000 rpm 离心1 min后，小心的吸出上清，将上清留存进行检测分析。
4、 洗杂
使用 5 CV的裂解液洗涤柱子，轻柔上下翻动混匀，1000 rpm 离心1 min后，小心的吸出上清，将上清留存进行检测分析。重复3-5次。如使用重力柱，则直接在重力柱中清洗，直接重力流干洗涤液。
5、 洗脱
加入3-5 CV的1×GSH洗脱缓冲液进行洗脱，室温孵育5 min，1000 rpm离心1 min或重力柱管收集洗脱液，可重复2-3次。
上述步骤洗脱结束后，用平衡液冲洗3 CV，然后用纯水冲洗5 CV，再用保护液冲洗2 CV，然后将介质置于2-8℃保存。
四、 样品检测与填料清洗
1、 SDS-PAGE 检测
将纯化过程中得到的样品(包括原始样品、流出组分、洗杂及洗脱组分等)利用 SDS-PAGE 进行检测，判定其纯化效果。
2、 填料清洗
在进行一定次数的使用后(具体使用次数和上样中的污染物有关)，需要对柱子进行清洗，以下提供一种方法：
用2 CV 6 M盐酸胍溶液进行清洗，立即用5 CV的纯水清洗。再用保护液冲洗2 CV，然后将介质置于2-8℃保存。
去除一些疏水性吸附造成的非特异性吸附物质：用3-4 CV的70%乙醇或2 CV的1% Triton X-100清洗，立即用5 CV的纯水清洗，再用保护液冲洗2 CV，然后将介质置于2-8℃保存。
注意事项
1、 请勿冷冻保存 GST亲和层析介质。
2、 GST亲和层析介质储存在含20%乙醇的1×PBS中，凝胶和保护液的体积比为1:1。
3、 GST亲和层析介质使用前一定要充分颠倒若干次，使琼脂糖珠混合均匀。
4、 本产品仅作科研用途。
5、 为了您的安全和健康，请穿实验服并佩戴一次性手套操作。
GST, or Glutathione S-Transferase, is expressed as a fusion with the target protein to generate a GST-tagged recombinant protein, also known as a GST-tagged protein or GST fusion protein. GST-tagged proteins bind specifically to GST Affinity Resin, while other proteins do not. Bound GST-tagged proteins can be eluted with excess reduced glutathione (GSH), enabling the separation and purification of the target protein. Since purification is performed under mild, native conditions, the target protein retains its biological activity, making it suitable for structural and functional studies, antibody preparation, protein‑protein interaction assays, and protein‑nucleic acid interaction studies. This kit supports purification of GST‑tagged proteins expressed in Escherichia coli, mammalian cells, insect cells, and baculovirus systems. GST fusion proteins often show high solubility in E. coli and help maintain the native activity of the target protein. Many eukaryotic proteins expressed in E. coli form inclusion bodies, but a significant portion can be expressed in soluble form when fused to GST, facilitating subsequent purification. If a site‑specific protease cleavage site, such as the recognition sequence for TEV Protease (rp155927), is inserted between the GST tag and the target protein, the GST tag can be removed using the corresponding protease. Fusing GST to the N‑terminus of the target protein generally preserves GST enzymatic activity and simplifies purification using GST Affinity Resin.
· Key component: GST Affinity Resin.
· Matrix: 4% cross‑linked agarose.
· Ligand: reduced glutathione, covalently coupled via a 12‑atom spacer arm.
· Binding capacity: >20 mg GST fusion protein per mL settled resin.
· Particle size: 45-165 μm.
· Maximum operating pressure: 0.1 MPa (1 bar).
The resin is supplied in 1×PBS containing 20% ethanol. A 10 mL slurry consists of 5 mL settled resin and 5 mL storage solution. The resin must be fully resuspended before pipetting. Actual binding capacity depends on the molecular weight of the target GST‑tagged protein: higher molecular weight generally results in higher binding capacity. This kit is sufficient for purification of up to 10 GST‑tagged proteins, with a typical yield of approximately 10 mg per protein.
Product Features
1. Rapid and Efficient: The kit uses high‑performance GST affinity resin with strong binding efficiency, shortening operation time and effectively preventing degradation or denaturation of the target protein during prolonged handling, fully preserving protein activity.
2. Convenient: All required reagents and empty gravity chromatography columns are provided, greatly simplifying purification of GST‑tagged proteins.
3. High Binding Capacity: 20 mg GST fusion protein can be bound per mL of settled resin.
4. High Specificity: Enables one‑step purification of glutathione S‑transferases, glutathione‑dependent proteins, and GST recombinant derivatives from various expression systems.
1 Sample Preparation
1.1 Soluble Protein Expressed in Bacteria or Yeast
1.1.1 Pick a single colony into medium containing the appropriate antibiotic. Induce expression with inducer at the recommended concentration and duration according to the vector instructions.
1.1.2 After expression, transfer the culture to a centrifuge bottle and centrifuge at 7000 rpm (7500 ×g) for 15 min to harvest cells. Resuspend the cell pellet in Native Lysis Buffer at a ratio of 1:10 (w/v). Optionally add PMSF to a final concentration of 1 mM or other protease inhibitors if the protein is prone to degradation, provided they do not interfere with binding to the resin. Lysozyme may be added as needed (not required if the host strain contains pLysS or pLysE).
1.1.3 Fully resuspend the cell pellet. If the cell suspension is concentrated, add 10 μg/mL RNase A and 5 μg/mL DNase I. Mix well, keep on ice, and lyse by sonication in an ice-water bath until the lysate becomes clear.
1.1.4 Transfer the lysate to a centrifuge tube and centrifuge at 10000 rpm (15000 ×g) at 4 °C for 20-30 min. Collect the supernatant, keep on ice, and proceed to purification.
1.2 Soluble Protein Expressed in Yeast, Insect, or Mammalian Cells
Transfer the cell culture to a centrifuge bottle and centrifuge at 5000 rpm (3800 ×g) for 10 min. Collect the supernatant. If the supernatant contains no EDTA, histidine, or reducing agents, it may be loaded directly. If EDTA, histidine, or reducing agents are present, dialyze against wash buffer before loading. For large volumes, concentrate by ammonium sulfate precipitation and dialyze against wash buffer before loading.
2 Buffer Preparation
2.1 10×GSH Stock Solution:
Dissolve the 184 mg GSH provided in the kit in 6 mL Elution Buffer and mix thoroughly.
Store the prepared 10×GSH solution at -20 °C, stable for at least 1 year.
2.2 1×GSH Elution Buffer:
Mix Elution Buffer and 10×GSH solution at a 9:1 ratio (e.g., 9 mL Elution Buffer + 1 mL 10×GSH). Since GSH is easily oxidized and inactivated, prepare fresh before use. The prepared 1×GSH Elution Buffer may be stored at 4 °C and used within 2 weeks.
3 Sample Purification
Before loading, samples are recommended to be centrifuged or filtered through a 0.22 μm or 0.45 μm filter to reduce impurities, improve purification efficiency, and prevent column clogging. Buffers are also recommended to be filtered through a 0.22 μm or 0.45 μm filter before use.
3.1 Gravity Column Purification
3.1.1 Column Packing
3.1.1.1 Take an empty gravity column, insert the bottom frit, rinse with purified water, and close the bottom outlet.
3.1.1.2 Mix the GST Affinity Resin thoroughly and pipette the slurry into the gravity column. The settled resin volume is approximately half the slurry volume (e.g., 1 mL slurry yields ~0.5 mL resin, with a maximum capacity of ~10 mg). Open the outlet to drain the storage solution.
3.1.1.3 Wash the resin with purified water. Drain completely and close the outlet.
3.1.1.4 Insert the top frit, ensuring no air gap between frit and resin and that the frit is level.
3.1.1.5 The packed column may be equilibrated immediately with wash buffer. If not used immediately, add storage solution and store at 4 °C.
3.1.2 Equilibration
Equilibrate the column with 2-5 CV Lysis Buffer (CV = settled resin volume; this definition applies throughout the protocol). Repeat 2-3 times.
3.1.3 Sample Loading
Apply the sample to the equilibrated column. Allow the sample to flow slowly to ensure sufficient binding. Collect the flow-through; reloading may increase binding efficiency. Keep samples on ice if the target protein is unstable.
3.1.4 Washing
Wash with 5-10 CV Lysis Buffer to remove non‑specifically bound proteins. Collect the wash fraction. During washing and subsequent elution, protein concentration in each wash and elution fraction can be rapidly monitored using the Bradford method to adjust the number of washing and elution steps as needed.
3.1.5 Elution
Elute the target protein with 5-10 CV 1×GSH Elution Buffer. Collect fractions every 1-5 CV and analyze individually to ensure complete elution and obtain highly pure, concentrated protein. After elution, wash with 3 CV equilibration buffer, 5 CV purified water, and 2 CV storage solution. Store the resin at 2-8 °C.
3.2 Batch Purification
3.2.1 Resin Preparation
Transfer an appropriate volume of GST Affinity Resin into a centrifuge tube. Centrifuge at 1000 rpm for 1 min and carefully remove the supernatant (use low speed to avoid damaging the resin). Alternatively, drain the storage solution in a gravity colum.
3.2.2 Resin Equilibration
Add 5 CV Lysis Buffer, mix gently by inversion, centrifuge at 1000 rpm for 1 min, and carefully remove the supernatant. Repeat at least twice. For gravity columns, wash and drain by gravity.
3.2.3 Binding
Add the prepared sample to the equilibrated resin. Seal the tube or column and mix by rotation. Incubate at room temperature for 30 min-2 h or at 2-8 °C for 2-4 h (longer if needed). Keep at 2-8 °C if the protein is unstable. Centrifuge at 1000 rpm for 1 min and retain the supernatant for analysis.
3.2.4 Washing
Wash with 5 CV Lysis Buffer, mix gently, centrifuge at 1000 rpm for 1 min, and retain the supernatant. Repeat 3-5 times. For gravity columns, wash and drain by gravity.
3.2.5 Elution
Elute with 3-5 CV 1×GSH Elution Buffer. Incubate at room temperature for 5 min. Centrifuge at 1000 rpm for 1 min or collect by gravity flow. Repeat 2-3 times. After elution, wash with 3 CV equilibration buffer, 5 CV purified water, and 2 CV storage solution. Store the resin at 2-8 °C.
4 Sample Detection and Resin Cleaning
4.1 SDS-PAGE
Analyze samples from the purification process (starting material, flow-through, wash, and elution fractions) by SDS-PAGE to evaluate purification efficiency.
4.2 Resin Cleaning
After repeated use, cleaning is required (frequency depends on sample contaminants).
General cleaning: Wash with 2 CV 6 M guanidine hydrochloride, then immediately with 5 CV purified water, then 2 CV storage solution. Store at 2-8 °C.
Removal of hydrophobic contaminants: Wash with 3-4 CV 70% ethanol or 2 CV 1% Triton X‑100, then immediately with 5 CV purified water, then 2 CV storage solution. Store at 2-8 °C.
Matters needing attention
1. Do not freeze the GST Affinity Resin.
2. GST Affinity Resin is supplied in 1×PBS containing 20% ethanol, with a 1:1 ratio of settled resin to storage solution.
3. The resin must be thoroughly resuspended by repeated inversion before use to ensure uniform agarose bead suspension.
4. This product is for research use only.
5. For your safety and health, wear a lab coat and disposable gloves during all experiments.
规格
别名
GST标签蛋白纯化试剂盒(pH 8.0)
英文别名
GST-Tag Protein Purification Kit (pH 8.0)
规格或纯度
BioReagent, 用于蛋白分析
英文名称
GST-Tag Protein Purification Kit (pH 8.0)
应用
Protein purification
储存与运输
储存条件
2-8°C储存,室温,禁止冷冻
运输条件
低温运输
稳定性与储存
各组分在相应的储存条件下保质期1年。
组分与储存
G15058023 | Components | 10T | Storage | Quantity Per Test |
G15058023A | GST亲和介质 | 10 mL | 2-8°C, Do not freeze | 1 mL |
G15058023B | 裂解缓冲液 | 180 mL | 2-8°C | 18 mL |
G15058023C | 洗脱缓冲液 | 60 mL | 2-8°C | 6 mL |
G15058023D | GSH | 184 mg | 2-8°C | 18.4 mg |
G15058023E | 层析空柱，3mL（盖，塞，膜） | 10 EA | Room Temperature | 1 EA |
技术文档
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技术文档和文章
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