GST Pull-Down磁珠

产品名称：Glutathione Magnetic Beads for GST Pull-Down Assay， GST Pull-Down 磁珠

货号： IF9302

品牌：Engibody

关键词：GST Pull-Down磁珠，用于Pull-Down实验中抓取GST融合蛋白与互作蛋白的复合物

The glutathione S-transferase (GST) pull-down technique has become an invaluable tool for the life scientist interested in protein chemistry.

The basic pull-down assay is an in vitro technique that consists of a GST-tagged bait protein (the

researcher’s protein of interest) that can be used to identify putative binding partner(s) (the prey). The bait protein, purified from an appropriate expression system (e.g., Escherichia coli or baculovirus-infected insect cells), is immobilized to glutathione beads. The bait serves as the secondary affinity support for confirming a previously suspected protein partner or for identifying new protein partners to the bait. Prey protein can be obtained from multiple sources, including previously purified proteins, cell lysates or in vitro transcription/translation reactions.

Protein:protein interactions can be visualized by SDS-PAGE and associated detection methods depending on the sensitivity requirements of the interacting proteins. These methods include coomassie, silver, zinc staining and Western blotting.

Tested applications

GST Pull-Down Assay.

Optimal amount should be determined by the end user.

Specificity

Binding GST-Tagged Fusion Protein.

Form

Magnetic Beads in Suspension (20% slurry), supplied in phosphate buffered saline (PBS) pH 7.4, with 0.02% sodium azide as preservatives.

Storage instruction

Store at 4°C. This product is stable for 12 months. Do not freeze, dry or centrifuge magnetic beads. This may cause irreversible aggregation and decreased binding capacity.

Bead Diameter:

40 µm

Concentration:

20% slurry

Binding Capacity

5 to10 mg GST fusion protein per 1 mL settled beads (5mL 20% slurry).

Protocols

Procedure Summary for GST Pull-Down Assay

I. Preparation of Bait Protein

Protocol 1: Bait from E. coli Expression Systems

Protocol 2: Bait from Insect Cell Expression Systems

Protocol 3: Bait from Previously Purified Protein

II. Immobilization of Bait Protein

A. Preparation of Glutathione beads

B. Immobilize Bait Protein

III. Preparation of Prey Protein

Protocol 1: Prey Protein from E. coli or Baculovirus-Infected Insect Cell Lysate

Protocol 2: Prey Protein from Mammalian Cell Lysate

Protocol 3: Prey from Previously Purified Protein

Protocol 4: Prey Protein from In vitro Transcription/Translation Reaction

IV. Capture of Prey Protein

V. Elution of Bait-Prey

VI. SDS PAGE Gel Analysis or WB Analysis

The glutathione S-transferase (GST)  pull down技术已成为对蛋白质化学感兴趣的生命科学家的宝贵工具。

Pull down技术是一种体外技术，由GST标记的诱饵蛋白（研究人员感兴趣的蛋白质），可用于鉴定推定的结合伴侣（猎物）。从合适的表达系统（例如大肠杆菌或杆状病毒感染的昆虫细胞）纯化的诱饵蛋白固定在GSH磁珠上。该诱饵蛋白用作第二亲和支持物，用于确认先前怀疑的蛋白质伴侣或识别诱饵的新蛋白质伴侣。Prey蛋白可以从多种来源获得，包括先前纯化的蛋白、细胞裂解物或体外转录/翻译反应。

蛋白质：蛋白质相互作用可通过SDS-PAGE和相关检测方法可视化，具体取决于相互作用蛋白质的敏感性要求。这些方法包括考马斯、银、锌染色和蛋白质印迹。

经过测试的应用

GST pull down assay

最佳用量应由最终用户确定。

特异性

结合GST标记的融合蛋白。

类型

悬浮液中的磁珠（20%浆料），在pH 7.4的磷酸盐缓冲盐水（PBS）中提供，含0.02%防腐剂。

存储说明

储存于4°C。该产品稳定12个月。请勿冷冻、干燥或离心磁珠。这可能导致不可逆聚集和结合能力降低。

磁珠直径：

40µm

浓度：

20%浆料

结合能力：

每1mL沉淀磁珠（5mL 20%浆料）5至10mg GST融合蛋白。

GST pull down 实验流程

一、 诱饵蛋白的制备

方案1：来自大肠杆菌表达系统的诱饵

方案2：来自昆虫细胞表达系统的诱饵

方案3：来自先前纯化蛋白的诱饵

二、诱饵蛋白的固定化

A、 GSH磁珠的准备

B、 固定化诱饵蛋白

三、 猎物蛋白的制备

方案1：来自大肠杆菌或杆状病毒感染的昆虫细胞裂解物的捕食蛋白

方案2：来自哺乳动物细胞裂解物的猎物蛋白

方案3：来自先前纯化的蛋白质的猎物

方案4：来自体外转录/翻译反应的猎物蛋白

四、 捕获猎物蛋白质

五、 诱饵蛋白的洗脱

六、 SDS PAGE凝胶分析或WB分析