產品名稱：GST tagged Protein Purification Kit

品牌：Engibody

貨號：P0007

簡介：GST蛋白純化試劑盒，用于GST標簽融合蛋白的富集純化。

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)瓊脂糖是一種高容量、高性能樹脂，用于從細胞裂解物中親和純化GST標記的融合蛋白。

高質量載體由glutathione (GSH)組成，glutathione (GSH)通過12原子間隔臂被其中心巰基固定到交聯的6%珠狀瓊脂糖上。使用glutathione (GSH)瓊脂糖珠純化GST融合蛋白已被廣泛記錄，并適用于各種規模和特定應用。無論目的是從過度表達的大腸桿菌裂解物中純化大量重組蛋白，還是研究涉及GST標記誘餌蛋白的蛋白質相互作用，Engibody公司的glutathione (GSH)瓊脂糖都適合這項任務。

帶有glutathione (GSH)樹脂的GST標記蛋白純化試劑盒允許快速親和純化GST標記的蛋白，用于批量、重力流和FPLC應用。該樹脂基于6%交聯瓊脂糖，glutathione (GSH)與樹脂共價結合。

該GST標記的融合蛋白純化試劑盒（P0007）包括一個重力柱、glutathione (GSH)瓊脂糖樹脂6FF、平衡緩沖液、洗滌緩沖液和洗脫緩沖液。

產品描述

GST標記蛋白純化試劑盒（glutathione (GSH)瓊脂糖珠6FF，可溶性蛋白）

試劑盒組分

•glutathione (GSH)瓊脂糖樹脂6FF，5 mL，類別：P3401，儲存于4°C

•重力柱

•GST標記蛋白純化用平衡緩沖液，100 mL，儲存于4°C

•GST標記蛋白純化用洗滌緩沖液，100 mL，儲存于4°C

•GST標記蛋白純化用洗脫緩沖液，100 mL，儲存于4°C

產品優點

•快速、一步純化GST標記蛋白

•溫和洗脫條件，保護蛋白質抗原性和功能

•glutathione (GSH)通過10個碳間隔臂連接到Beads 6 Fast Flow

•整套試劑盒包括裂解緩沖液、洗滌緩沖液、洗脫緩沖液、樹脂和柱

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標簽融合蛋白純化步驟

1.細胞提取物的制備

1). 通過在4°C下以3000 g離心10分鐘收集細胞，移除并丟棄上清液。

2). 每50 mL培養物將細胞顆粒重新懸浮在3 mL冰冷的1×PBS緩沖液中，并在4°C下以3000 g離心10分鐘。取出并丟棄上清液。

3). 將細胞顆粒在-80°C下冷凍1小時（這也是一個方便的停止點，稍后可以繼續操作）。

4). 將細胞沉淀在冰上解凍，并將細胞重新懸浮在3 mL冰冷的1×PBS緩沖液中/50 mL培養物中。如果需要，添加適當的添加劑，如非離子洗滌劑（NP-40）或蛋白酶抑制劑。

5). 通過在冰上短暫的超聲波脈沖破壞細胞，直到樣品不再粘稠。

6). 在4°C下12000 g離心10分鐘，并小心地將上清液（可溶性部分）轉移到干凈且預冷的管中，并在每50 mL培養物中用3 mL冰冷的1×PBS緩沖液重新懸浮顆粒（不溶性部分）。

7). 從可溶性和不溶性級分中等分10μL樣品進行SDS-PAGE分析（通過加入等量的2X SDS樣品緩沖液（125mM Tris HCl，pH 6.8，4%w/v SDS，20%甘油，100mM DTT，0.02%w/v溴酚藍），煮沸5分鐘并運行SDS-PAGE以確定GST融合蛋白的量和溶解度）。

注：

1). GST或GST融合蛋白與glutathione (GSH)樹脂的結合不受1%Triton X-100、1%Tween-20、1%CTAB、10mM DTT、0.03%SDS或0.1%NP-40的影響。這些化學物質可用于減少非特異性結合。

2). 如果目標GST融合蛋白形成包涵體（不溶性蛋白），則必須在純化前將包涵體適當溶解并重新折疊。

2.重組GST融合蛋白的純化

1). 填充柱并平衡glutathione (GSH)樹脂。

將重力柱固定在鐵架上，依次將下部玻璃料、GSH樹脂（1mL-2.5mL沉淀樹脂）和上部玻璃料放入空柱中。

向柱管中加入5ml平衡緩沖液，平衡樹脂，排水后，重復2次，總共使用15ml平衡緩沖液。

2). 添加GST標記的蛋白細胞裂解物樣品。

將處理過的蛋白質樣品加入柱管中，收集流動，并使用SDS-PACE分析目標蛋白質和樹脂的結合。當問題發生時，找到解決問題的方法更方便。

注意：通常1 mL沉淀樹脂（2 mL 50%漿料）可以結合超過25 mg GST蛋白（26kD）。

3). 用洗滌緩沖液洗滌GST標記的蛋白/glutathione (GSH)樹脂復合物。

向柱管中加入5ml洗滌緩沖液以去除非特異性結合蛋白，收集洗滌級分，重復5次，總共使用30ml洗滌緩沖。

4). 通過洗脫緩沖液洗脫GST標記的蛋白質。

使用15-30ml洗脫緩沖液洗脫目標蛋白，分段收集（一管約為5ml），并通過運行SDS-PAGE分別分析所有切片，以確認目標蛋白的存在，以確保洗脫所有結合蛋白，獲得高純度和高濃度蛋白。使用280nm處的吸光度讀數監測融合蛋白的洗脫。通過在4°C下用所選緩沖液透析或使用G15葡聚糖脫鹽柱除去游離glutathione (GSH)。

5). 重新平衡glutathione (GSH)樹脂（位于重力柱中）。

交替使用5 ml平衡緩沖液和5 ml去離子水重新平衡GSH樹脂，重復2次，最后用5 ml 20%乙醇平衡GSH，重復一次，然后儲存在等體積的20%乙醇中，并在2-8度下儲存，防止樹脂受到細菌污染。

3.SDS-PAGE試驗

在SDS-PAGE測試中，使用純化過程中的級分（包括流動組分、洗滌組分和洗脫組分）和全細胞提取物作為樣品，以便評估純化效果。

4.樹脂清洗

GSH樹脂可以重復使用幾次而無需再生，但隨著非特異性結合蛋白和蛋白聚集的增加，流速和結合能力降低，樹脂需要清洗。

為了去除一些沉淀或變性物質，建議采用以下方法：

用2柱床體積的6M鹽酸胍溶液洗滌，然后立即用5柱床體積pH 7.4的PBS洗滌。

為了消除疏水吸附引起的非特異性吸附劑，建議采用以下方法：

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

僅用于科研，不作用人體