产品名称：GST tagged Protein Purification Kit

品牌：Engibody

货号：P0007

简介：GST蛋白纯化试剂盒，用于GST标签融合蛋白的富集纯化。

Glutathione Agarose is a high-capacity, high-performance resin for affinity purification of GST-tagged fusion proteins from cellular lysates.

The high-quality support consists of glutathione that has been immobilized by its central sulfhydryl group via a 12-atom spacer arm to crosslinked 6% beaded agarose. Purification of GST-fusion proteins using glutathione (GSH) agarose beads is well documented and adaptable to a variety of scales, column formats and specific applications. Whether the purpose is to purify large amounts of recombinant protein from over-expressing E. coli lysates or to investigate protein interactions involving GST-tagged bait proteins, Engibody Glutathione Agarose is suitable for the task.

GST-tagged protein purification kit with Glutathione Resin allows rapid affinity purification of GST-tagged proteins for batch, gravity flow, and FPLC applications. The resin is based on 6% cross-linked agarose, with glutathione covalently bound to the resin.

This GST tagged Fusion Protein Purification Kit (P0007) includes one gravity column, Glutathione Agarose Resin 6FF, Equilibration Buffer, Wash Buffer and Elution Buffer.

Description

GST tagged Protein Purification Kit (Glutathione Agarose Beads 6FF, Soluble Protein)

Components

• Glutathione Agarose Resin 6FF, 5 mL, Cat: P3401, store at 4°C

• Gravity Column

• Equilibration Buffer for GST tagged Protein Purification, 100 mL, store at 4°C

• Wash Buffer for GST tagged Protein Purification, 100 mL, store at 4°C

• Elution Buffer for GST tagged Protein Purification, 100 mL, store at 4°C

Highlights

• Fast, one-step purification of GST-tagged proteins

• Mild elution conditions preserving protein antigenicity and function

• Glutathione (GSH) coupled to Beads 6 Fast Flow via a 10-carbon spacer arm

• Complete kit—includes lysis buffer, wash buffer, and elution buffer, resin and column

glutathione (GSH)琼脂糖是一种高容量、高性能树脂，用于从细胞裂解物中亲和纯化GST标记的融合蛋白。

高质量载体由glutathione (GSH)组成，glutathione (GSH)通过12原子间隔臂被其中心巯基固定到交联的6%珠状琼脂糖上。使用glutathione (GSH)琼脂糖珠纯化GST融合蛋白已被广泛记录，并适用于各种规模和特定应用。无论目的是从过度表达的大肠杆菌裂解物中纯化大量重组蛋白，还是研究涉及GST标记诱饵蛋白的蛋白质相互作用，Engibody公司的glutathione (GSH)琼脂糖都适合这项任务。

带有glutathione (GSH)树脂的GST标记蛋白纯化试剂盒允许快速亲和纯化GST标记的蛋白，用于批量、重力流和FPLC应用。该树脂基于6%交联琼脂糖，glutathione (GSH)与树脂共价结合。

该GST标记的融合蛋白纯化试剂盒（P0007）包括一个重力柱、glutathione (GSH)琼脂糖树脂6FF、平衡缓冲液、洗涤缓冲液和洗脱缓冲液。

产品描述

GST标记蛋白纯化试剂盒（glutathione (GSH)琼脂糖珠6FF，可溶性蛋白）

试剂盒组分

•glutathione (GSH)琼脂糖树脂6FF，5 mL，类别：P3401，储存于4°C

•重力柱

•GST标记蛋白纯化用平衡缓冲液，100 mL，储存于4°C

•GST标记蛋白纯化用洗涤缓冲液，100 mL，储存于4°C

•GST标记蛋白纯化用洗脱缓冲液，100 mL，储存于4°C

产品优点

•快速、一步纯化GST标记蛋白

•温和洗脱条件，保护蛋白质抗原性和功能

•glutathione (GSH)通过10个碳间隔臂连接到Beads 6 Fast Flow

•整套试剂盒包括裂解缓冲液、洗涤缓冲液、洗脱缓冲液、树脂和柱

Purification Protocol

1. Preparation of Cell Extract

1). Harvest cells by centrifugation at 3,000 g at 4°C for 10 min, remove and discard the supernatant.

2). Resuspend the cell pellet in 3 mL ice-cold 1×PBS buffer per 50 mL culture and centrifuge at 3,000 g at 4°C for 10 min. Remove and discard the supernatant.

3). Freeze the cell pellet at -80°C for 1 hour (This is also a convenient point to stop and one can continue the procedure later).

4). Thaw cell pellet on ice and resuspend cells in 3 mL of ice-cold 1×PBS buffer per 50 mL culture. If desired, add appropriate additives, such as non-ionic detergents (NP-40) or protease inhibitors.

5). Disrupt cells by brief pulses of sonication on ice until the sample is no longer viscous.

6). Centrifuge at 12,000 g at 4°C for 10 min and carefully transfer the supernatant (soluble fraction) to a clean and pre-chilled tube and resuspend pellet (insoluble fraction) with 3 mL of ice-cold 1×PBS buffer per 50 mL culture.

7). Aliquot 10μL samples from both soluble and insoluble fractions for SDS-PAGE analysis (by adding equal volume of 2X SDS Sample Buffer (125mM Tris-HCl, pH 6.8, 4% w/v SDS, 20% glycerol, 100mM DTT, 0.02% w/v bromophenol blue), boiling for 5 min and running SDS-PAGE to determine the amount and solubility of the GST-fusion protein).

Note:

1). The binding of GST or GST-fusion protein to Glutathione Resin is not affected by 1% Triton X-100, 1% Tween-20, 1% CTAB, 10 mM DTT, 0.03% SDS, or 0.1% NP-40. These chemicals may be used to reduce non-specific binding.

2). If the target GST-fusion protein forms inclusion body (insoluble protein), the inclusion body has to be properly solubilized and refolded prior to purification.

2. Purification of Recombinant GST-Fusion Protein

1). Pack column and Equilibrate Glutathione (GSH) Resin.

Fix the Gravity Column on the iron frame, put the lower frit, GSH resin (1 mL - 2.5mL settled resin) and upper frit into the empty column in turn.

Add 5ml of Equilibration Buffer to the column tube, equilibrate the resin, and after draining, repeat 2 more times, using 15ml of Equilibration Buffer in total.

2). Add GST tagged protein cell lysate sample.

Add the processed protein sample to the column tube, collect the flow through, and use it to analyze the binding of target protein and resin by SDS-PACE. When a problem occurs, it is more convenient to find a solution to the problem.

Attention: Usually 1 mL settled resin (2 mL 50% slurry) can bind more than 25 mg GST protein(26kD).

3). Wash GST tagged protein/Glutathione (GSH) Resin complex by wash buffer.

Add 5 ml Wash Buffer into the column tube in order to remove non-specific binding protein, collect wash fractions, repeat 5 times, use 30 ml Wash Buffer in total.

4). Elute GST tagged protein by elution buffer.

Use 15-30 ml of Elution Buffer to elute the target protein, collect in sections (one tube is about 5 ml), and respectively analyze all the sections by running SDS-PAGE to confirm the presence of the target protein, so as to ensure that all bound proteins are eluted, high-purity and high-concentration proteins can be obtained. Monitor elution of the fusion protein using absorbance readings at 280 nm. Remove free glutathione by dialysis at 4°C against a buffer of choice or by using a G15 Sephadex desalt column.

5). Re-Equilibrate Glutathione (GSH) Resin (which is in gravity column).

Use 5 ml Equilibration Buffer and 5 ml deionized water alternately to re-equilibrate the GSH resin, repeat 2 times, and finally equilibrate the GSH resin with 5 ml 20% ethanol, repeat once, then store in an equal volume of 20% ethanol, and Store at 2-8 degrees, and prevent resin from bacteria pollution.

3. SDS-PAGE Test

Use the fractions from purification process (including flow through, wash fractions and elution fractions) and whole cell extract as samples in SDS-PAGE test, so that we can evaluate the purification effect.

4. Resin Cleaning

GSH resin can be reused several times without regeneration, but with the increase of non-specifically bound proteins and protein aggregation, the flow rate and binding capacity are reduced, and the resin needs to be washed.

To remove some precipitated or denatured substances, the following method is recommended:

Wash with 2 column bed volumes of 6M guanidine hydrochloride solution, followed immediately by 5 column bed volumes of PBS, pH 7.4.

To Eliminate non-specific adsorbents caused by hydrophobic adsorption:

Wash with 3-4 column bed volumes of 70% ethanol or 2 column bed volumes of 1% Triton X-100, followed immediately by 5 column bed volumes of PBS, pH 7.4.

GST标签融合蛋白纯化步骤

1.细胞提取物的制备

1). 通过在4°C下以3000 g离心10分钟收集细胞，移除并丢弃上清液。

2). 每50 mL培养物将细胞颗粒重新悬浮在3 mL冰冷的1×PBS缓冲液中，并在4°C下以3000 g离心10分钟。取出并丢弃上清液。

3). 将细胞颗粒在-80°C下冷冻1小时（这也是一个方便的停止点，稍后可以继续操作）。

4). 将细胞沉淀在冰上解冻，并将细胞重新悬浮在3 mL冰冷的1×PBS缓冲液中/50 mL培养物中。如果需要，添加适当的添加剂，如非离子洗涤剂（NP-40）或蛋白酶抑制剂。

5). 通过在冰上短暂的超声波脉冲破坏细胞，直到样品不再粘稠。

6). 在4°C下12000 g离心10分钟，并小心地将上清液（可溶性部分）转移到干净且预冷的管中，并在每50 mL培养物中用3 mL冰冷的1×PBS缓冲液重新悬浮颗粒（不溶性部分）。

7). 从可溶性和不溶性级分中等分10μL样品进行SDS-PAGE分析（通过加入等量的2X SDS样品缓冲液（125mM Tris HCl，pH 6.8，4%w/v SDS，20%甘油，100mM DTT，0.02%w/v溴酚蓝），煮沸5分钟并运行SDS-PAGE以确定GST融合蛋白的量和溶解度）。

注：

1). GST或GST融合蛋白与glutathione (GSH)树脂的结合不受1%Triton X-100、1%Tween-20、1%CTAB、10mM DTT、0.03%SDS或0.1%NP-40的影响。这些化学物质可用于减少非特异性结合。

2). 如果目标GST融合蛋白形成包涵体（不溶性蛋白），则必须在纯化前将包涵体适当溶解并重新折叠。

2.重组GST融合蛋白的纯化

1). 填充柱并平衡glutathione (GSH)树脂。

将重力柱固定在铁架上，依次将下部玻璃料、GSH树脂（1mL-2.5mL沉淀树脂）和上部玻璃料放入空柱中。

向柱管中加入5ml平衡缓冲液，平衡树脂，排水后，重复2次，总共使用15ml平衡缓冲液。

2). 添加GST标记的蛋白细胞裂解物样品。

将处理过的蛋白质样品加入柱管中，收集流动，并使用SDS-PACE分析目标蛋白质和树脂的结合。当问题发生时，找到解决问题的方法更方便。

注意：通常1 mL沉淀树脂（2 mL 50%浆料）可以结合超过25 mg GST蛋白（26kD）。

3). 用洗涤缓冲液洗涤GST标记的蛋白/glutathione (GSH)树脂复合物。

向柱管中加入5ml洗涤缓冲液以去除非特异性结合蛋白，收集洗涤级分，重复5次，总共使用30ml洗涤缓冲。

4). 通过洗脱缓冲液洗脱GST标记的蛋白质。

使用15-30ml洗脱缓冲液洗脱目标蛋白，分段收集（一管约为5ml），并通过运行SDS-PAGE分别分析所有切片，以确认目标蛋白的存在，以确保洗脱所有结合蛋白，获得高纯度和高浓度蛋白。使用280nm处的吸光度读数监测融合蛋白的洗脱。通过在4°C下用所选缓冲液透析或使用G15葡聚糖脱盐柱除去游离glutathione (GSH)。

5). 重新平衡glutathione (GSH)树脂（位于重力柱中）。

交替使用5 ml平衡缓冲液和5 ml去离子水重新平衡GSH树脂，重复2次，最后用5 ml 20%乙醇平衡GSH，重复一次，然后储存在等体积的20%乙醇中，并在2-8度下储存，防止树脂受到细菌污染。

3.SDS-PAGE试验

在SDS-PAGE测试中，使用纯化过程中的级分（包括流动组分、洗涤组分和洗脱组分）和全细胞提取物作为样品，以便评估纯化效果。

4.树脂清洗

GSH树脂可以重复使用几次而无需再生，但随着非特异性结合蛋白和蛋白聚集的增加，流速和结合能力降低，树脂需要清洗。

为了去除一些沉淀或变性物质，建议采用以下方法：

用2柱床体积的6M盐酸胍溶液洗涤，然后立即用5柱床体积pH 7.4的PBS洗涤。

为了消除疏水吸附引起的非特异性吸附剂，建议采用以下方法：

用3-4柱床体积70%乙醇或2柱床体积1%Triton X-100洗涤，然后立即用5柱床体积PBS（pH 7.4）洗涤。

仅用于科研，不作用人体