GST Pull-Down 实 验 基 于 谷 胱 甘 肽-S- 转 移 酶 蛋 白 （ GST ， glutathione-S-transferase）与谷胱甘肽（Glutathione，GSH）之间发生的可逆 的特异性结 合。通过GST标记的诱饵蛋白分离与纯化对应的未知捕获蛋白。 简单的实验原理是：将 GSH固定于琼脂糖凝胶上，形成GSH-琼脂糖凝胶；将诱饵蛋白与GST融合表达，获得的 GST-诱饵蛋白融合蛋白可与GSH-琼脂 糖凝胶结合；若环境中存在与诱饵蛋白互做的捕获 蛋白，则会形成“琼脂糖凝 胶-GSH-GST-诱饵蛋白-捕获蛋白”复合物，那么与诱饵蛋白互做 的捕获蛋白即可被分离并检测。 本产品由高品质的还原型谷胱甘肽（GSH）与琼脂糖凝胶共价偶联制成，具有高载量， 操作迅速便捷，特异性强和可特异性地与细胞或微生物裂解液等中含有GST标签的蛋白结 合等特点。具备操作简便、结合能力强、适用范围广等优点。
注意事项：
1. 本产品仅限于专业人员的科学研究使用。
2. 洗脱缓冲液(需添加GSH)添加GSH后，含有的GSH易被氧化，建议尽量现配现用。
3. 除非另有说明，所有操作建议于4°C下进行, 以尽量减少蛋白降解的可能性。若细胞裂解不够完全可在使用裂解液之后使用超声裂解细胞。
4. 谷胱甘肽琼脂糖树脂 填料应保存在储存溶液中，防止干燥，使用前请充分混匀。即颠倒若干次使凝胶混合均匀，混匀操作须轻柔，不宜剧烈涡旋震荡等，避免抗体变性等。
5. 经煮沸后的填料失去结合能力，经煮沸的填料不应再次使用。
6. 试剂用量可按实验实际进行取用，建议通过预实验进行免疫沉淀的验证或对裂解条件进行一定的调整。
7. 请注意安全事项，遵守实验室试剂操作规范操作。
8. 本试剂盒内包含的裂解液已包含蛋白酶抑制剂，如果有特殊需求，可考虑选择其它适当的抑制剂混合物。
9. 蛋白样品收集后宜尽快完成纯化工作，并应始终放置在4ºC或冰浴，以减缓蛋白降解或变性。
10. 在免疫沉淀或纯化时，建议设置阳性和阴性对照组。
G1511148 | 组份 | 5T | 25T | 储存温度 | 单次测试使用量 |
G1511148A | 谷胱甘肽琼脂糖树脂 | 0.2 mL | 1 mL | 2-8°C | 40 μL |
G1511148B | 1×裂解液 | 5 mL | 25 mL | 2-8°C | 每 250 μg 样品150 μL |
G1511148C | 谷胱甘肽 (还原型) | 0.2 g | 1 g | -20°C | 按需 |
G1511148D | 10×PBS | 10 mL | 50mL | RT | 0.5 mL |
G1511148E | 蛋白酶抑制剂(100×) | 0.05 mL | 0.25 mL | -20°C | 按需 |
使用方法 (除非另有说明，所有操作建议于4℃下进行)
1. 试剂准备
额外所需材料：
(1) 自备试剂：
a) 一抗：GST tag 抗体。
b) 二抗：Goat Anti-Mouse IgG H&L (HRP)、Goat Anti-Rabbit IgG H&L (HRP)。
c) 其它试剂： TBST、 电泳液、转膜液、 还原型 SDS-PAGE 凝胶以及蛋白互作所用诱饵蛋白、靶蛋白。
(2) 所需仪器：电泳仪、转膜仪、成像仪。
以上试剂若有需求，可以订购阿拉丁的 GST Tag Mouse mAb (Ab155828)、Recombinant GST tag Antibody (Ab106638)、Goat Anti-Rabbit IgG H&L (HRP) ( Ab176443)、Goat Anti-Mouse IgG H&L (HRP) (Ab179001)
2.溶液配制
可使用试剂盒准备的缓冲液，也可根据实际情况配制不同的缓冲液体系。所有缓冲液在使用前建议用0.22 μm或0.45 μm滤膜过滤，缓冲液建议4°C保存，若试剂浑浊，请立即丢弃。
(1) 洗脱液配制： 将GSH粉末，用分析天平称取3.1 mg GSH，加入到1 mL 1×PBS中稀释成终浓度10 mM谷胱甘肽洗脱缓冲液(现用现配)，然后用NaOH调至中性。
(2) 1×裂解液配制： 按照每50-100万细胞使用100-200 μL含抑制剂裂解液用于裂解。将裂解液与蛋白酶抑制剂 (100×)按照100:1的比例混合。例1 mL的裂解液中加入10 μL 蛋白酶抑制剂(100×)，即得1 mL含抑制剂裂解液。配制好的含抑制剂裂解液宜放置在冰浴或4 ºC暂存。
注1：含抑制剂裂解液宜现用现配，不宜配制后冻存并留作后续使用。
注2：该裂解液适用于哺乳动物细胞。
(3)1×PBS配制：按照 9:1 的比例用去离子水将 10×PBS稀释待用。例如：1mL 10×PBS 加 入 9 mL 去离子水，混匀后即为 1×PBS 。
(4) 凝胶漂洗。 轻轻重悬谷胱甘肽琼脂糖树脂，尽量形成均匀的凝胶悬浊液，按照通常每 40 μL混合均匀的凝胶悬浊液(以下亲和沉淀步骤中都以每样加入40 μL凝胶悬浊液为例)，取适量 GST标签蛋白纯化琼脂糖凝胶至一洁净离心管中，加入0.5 mL1×PBS，1250×g在4℃离心30秒，弃上清，重复此步骤2次 。
注：使用大孔径吸头(如用剪刀剪去部分吸头)吸取凝胶悬浊液会比较方便。
3. 诱饵蛋白的准备
(1) 对于对于大肠杆菌表达的GST标签融合蛋白 :
按照标准方案培养并转化大肠杆菌。取 5 mL 经 IPTG 诱导的大肠杆菌培养液，转移至无菌离心管中。5000×g 离心 5 分钟，弃去培养液上清。按每 5 mL 初始菌液体积加入 1 mL PBS 缓冲液重悬菌体沉淀，用移液枪吹打或涡旋混匀。取 1 mL 细胞悬液转移至 1.5 mL 微量离心管中。5000×g 离心 5 分钟，弃去上清。按每 5 mL 初始菌液体积加入 200 μL 预冷 PBS 缓冲液重悬沉淀，用移液枪吹打或涡旋混匀。可按需加入蛋白酶抑制剂；为获得最佳效果，制备细胞裂解液时建议使用蛋白酶抑制剂混合物。按每 5 mL 初始菌液体积加入 200 μL 亲和沉淀裂解缓冲液，立即颠倒离心管至充分混匀。冰上孵育约 30 分钟，期间定期颠倒离心管。12000×g 离心 5 分钟，使大肠杆菌粗裂解液澄清。将上清转移至另一支微量离心管，冰上保存，标记为诱饵蛋白裂解液。
(2) 对于分泌型GST标签融合蛋白：
收集培养基上清，检测诱饵蛋白浓度。如果诱饵蛋白质浓度较高，建议用1×PBS稀释至蛋白质终浓度为500 μg/mL，以备后续实验。
(3) 对于已经纯化好的GST标签融合蛋白:
检测诱饵蛋白浓度。如果诱饵蛋白质浓度较高，建议用1×PBS稀释至蛋白质终浓度为500 μg/mL，以备后续实验。
注：如果诱饵蛋白中含有GSH，需要先通过透析或者超滤的方式去除，才可以进行后续实验步骤。
4. Pull-Down 检测
1) 诱饵蛋白固定
(1) 将步骤3得到的诱饵蛋白加入预洗好的凝胶中，轻柔混匀，在4℃摇床上孵育3 h。此处需保留部分诱饵蛋白样品，作为input对照，以备后续检测使用。
(2) 离心分离。孵育结束后，1250×g在4℃离心30秒，将上清转移至新的离心管中，留样备检。
(3) 将离心管中的复合物转移到离心柱中，1250×g在4℃离心30秒，转移离心液至新的离心管中，标记为诱饵蛋白流穿液，以备后续检测使用。
注：对于细胞或细菌裂解液，加入至少500 µLGST标记的融合蛋白裂解液。对于已纯化好的GST标签融合蛋白，使用足够的体积以确保添加约100-150 µg的诱饵蛋白。
(4) 向离心柱中加入500 μL1×PBS，倒置几次温和混匀，清洗凝胶，1250×g在4℃离心30秒，弃离心液。重复4次。得到诱饵蛋白-凝胶复合物。
2) 铺获靶蛋白
(1) 向诱饵蛋白-凝胶复合物中，加入800 µL的准备好的捕获蛋白样品。在4°C的摇床上轻轻摇晃至少3小时或孵育过夜。此处需保留部分捕获蛋白样品，作为捕获蛋白input，以备后续检测使用。
注：为保证诱饵蛋白和捕获蛋白充分结合，可能需要更长的孵育时间，可根据实验情况调整。
(2) 孵育结束后，1250×g在4℃离心30秒，转移离心液至新的离心管中，标记为捕获蛋白流穿液，以备后续检测使用。
(3) 向离心柱加入500 μL1×PBS，倒置几次温和混匀，清洗凝胶，1250×g在4℃离心30秒，弃离心液。重复4次。得到诱饵-捕获蛋白-凝胶复合物。
5. 洗脱诱饵-捕获蛋白：根据标签蛋白的特点及后续实验要求，可以选择如下2种方法之一进行洗脱。
(1) 非变性洗脱法
吸取100 µL配制好的GST洗脱液加入到清洗好后的填料中，充分混匀填料与洗脱液后，进行离心并收集洗脱液。如洗脱效果不佳，则可提高谷胱甘肽浓度或多次洗脱。
(2) SDS-PAGE 上样缓冲液洗脱
每40 µL原始凝胶体积加入50uL 的1xPBS重悬凝胶，再加入50 µL的2×SDS Loading Buffer，轻摇摇晃混匀后95-100℃高温处理5-10分钟。1250×g在4℃离心30秒，取上清进行SDS-PAGE 电泳或进行Western 检测。
注: Western 检测的上样量建议控制在 20 µL 以内，剩余的样本可冻存于−20℃保存。
The GST Pull-Down assay is based on the reversible, specific interaction between glutathione-S-transferase (GST) and glutathione (GSH). It is used to isolate and purify unknown prey proteins using a GST-tagged bait protein. The basic principle is as follows: GSH is immobilized onto Agarose beads, forming GSH-Agarose beads. The bait protein is expressed as a fusion protein with GST, and the resulting GST−bait fusion protein can bind to the GSH-Agarose beads. If prey proteins that interact with the bait protein are present in the system, an "Agarose beads−GSH−GST-bait protein-prey protein" complex will form. This allows for the isolation and subsequent detection of the prey proteins that interact with the bait protein.
This product is manufactured by covalently coupling high−quality reduced glutathione (GSH) to Agarose beads. It features high binding capacity, rapid and convenient operation, and strong specificity. It can specifically bind to proteins containing a GST tag from sources such as cell or microbial lysates. The product offers the advantages of easy operation, high binding capability, and broad applicability.
Precautions
1. This product is intended for scientific research use by professionals only.
2. After adding GSH to the elution buffer (GSH required), the GSH contained is prone to oxidation. It is recommended to prepare the buffer fresh immediately before use .
3. Unless otherwise specified, all steps are recommended to be performed at 4°C to minimize potential protein degradation. If cell lysis is insufficient, sonication may be used after adding the lysis buffer.
4. GST-tag Purification Resin should be stored in the storage solution to prevent drying. Before use, mix thoroughly by gently inverting the tube several times to ensure uniform suspension of the beads. Avoid vigorous vortex or shaking to prevent antibody denaturation.
5. Agarose Resin that have been boiled lose their binding capacity and should not be reused.
6. Reagent volumes can be adjusted according to experimental needs. It is recommended to perform preliminary experiments to validate immunoprecipitation conditions or optimize lysis parameters.
7. Please follow safety guidelines and adhere to laboratory reagent handling protocols.
8. The lysis buffer included in this kit already contains protease inhibitors. If specific needs arise, other appropriate inhibitor cocktails can be used.
9. After harvesting, protein samples should be purified as soon as possible and kept at 4 °C or on ice at all times to minimize protein degradation and denaturation
10. It is recommended to include positive and negative control groups during immunoprecipitation or purification procedures.
G1511148 | Components | 5T | 25T | Storage Temperature | Quantity Per Test |
G1511148A | GST-tag Purification Resin | 0.2 mL | 1 mL | 2-8°C | 40 μL |
G1511148B | 1xLysis Buffer | 5 mL | 25 mL | 2-8°C | 150 μL per 250 μg sample |
G1511148C | Glutathione (Reduced, GSH) | 0.2 g | 1 g | -20°C | On request |
G1511148D | 10×BS | 10 mL | 50 mL | RT | 0.5 mL |
G1511148E | Protease Inhibitor Cocktail(100x) | 0.05 mL | 0.25 mL | -20°C | On request |
Procedure (Unless otherwise specified, all procedures should be performed at 4°C)
1.Reagent Preparation
Additional Required Materials:
(1) Reagents to be prepared by the user:
a) Primary Antibody: GST tag antibody.
b) Secondary Antibodies: Goat Anti-Mouse IgG H&L (HRP), Goat Anti-Rabbit IgG H&L (HRP).
c) Other Reagents: TBST, running buffer, transfer buffer, reducing SDS‑PAGE gel, as well as the bait protein and target protein used for protein‑protein interaction assays .
(2) Required Equipment:
Electrophoresis Apparatus, Transfer Apparatus, Imaging System
The above reagents, if required, can be ordered from Aladdin: GST Tag Mouse mAb (Ab155828), Recombinant GST tag Antibody (Ab106638), Goat Anti-Rabbit IgG H&L (HRP) (Ab176443), Goat Anti-Mouse IgG H&L (HRP)(Ab179001).
2. Solution Preparation
You may use the buffers provided in the kit, or prepare different buffer systems according to actual needs. It is recommended to filter all buffers through a 0.22 μm or 0.45 μm membrane filter before use. Buffers should be stored at 4°C. If any reagent appears cloudy, discard it immediately.
(1) Prepare an appropriate amount of inhibitor-containing lysis buffer based on the proportion of using 100-200 μL of inhibitor-containing lysis buffer for lysis and 300−600 μL of inhibitor-containing lysis buffer for washing per 0.5-1 million cells. Mix Lysis Buffer and Protease Inhibitor Cocktail (100×) at a ratio of 100:1. For example, add 10 μL of Protease Inhibitor Cocktail (100×) to 1 ml of lysis Buffer to obtain 1 ml of inhibitor-containing lysis buffer (Lysis Buffer with Protease Inhibitor Cocktail). The prepared inhibitor-containing lysis buffer should be placed on ice or at 4°C .
Note: The inhibitor-containing lysis buffer should be prepared fresh before use and should not be frozen and stored for later applications.
(2) This kit provides glutathione (GSH) powder. Weigh out 3.1 mg of the glutathione (GSH)powder using an analytical balance and add it to 1 mL of 1×PBS to prepare a glutathione elution buffer with a final concentration of 10 mM (prepare fresh before use). Then adjust the pH to neutral using NaOH.
(3) Preparation of 10×PBS: Dilute the 10×PBS with deionized water at a 9:1 ratio. For example, add 9 mL of deionized water to 1 mL of 10×PBS, and mix well to obtain the 1×PBS.
(4) GST-tag Purification Resin Washing: Gently resuspend the GST-tag Purification Resin to form a homogeneous gel suspension. Using 40 μL of well-mixed gel suspension per sample (all subsequent affinity precipitation steps use 40 μL gel suspension per sample as an example), transfer an appropriate amount of GST-tag protein purification agarose gel to a clean centrifuge tube. Add 0.5 mL of 1×PBS, centrifuge at 1250×g for 30 seconds at 4°C, discard the supernatant, and repeat this step twice.
Note: It is more convenient to aspirate the gel suspension using a wide-bore tip (e.g., by cutting off the end of a pipette tip with scissors).
3. Preparation of Bait Protein Samples (Note: Perform all sample lysis steps at 4°C or on ice)
(1)For GST-tagged fusion protein expressed in Escherichia coli:
Grow and transform E. coli according to standard protocols. Transfer 5 mL of IPTG-induced E. coli culture to a sterile centrifuge tube. Centrifuge at 5000×g for 5 minutes and discard the culture supernatant. Resuspend the pellet in 1 mL of PBS buffer per 5 mL of original culture volume, and mix by pipetting or vortex. Transfer 1 mL of cell suspension to a 1.5 mL microcentrifuge tube. Centrifuge at 5000×g for 5 minutes and discard the supernatant. Resuspend the pellet in 200 μL of ice-cold PBS buffer per 5 mL of original culture volume, and mix by pipetting or vortex. Protease inhibitors may be added as desired; for optimal results, use a protease inhibitor cocktail during cell lysate preparation. Add 200 μL of pull-down lysis buffer per 5 mL of original culture volume, and immediately invert the tube until thoroughly mixed. Incubate on ice for approximately 30 minutes, inverting the tube periodically. Centrifuge at 12000×g for 5 minutes to clarify the crude E. coli lysate. Transfer the supernatant to a new microcentrifuge tube, store on ice, and label as “bait lysate”.
(2) For secreted GST-tagged fusion protein:
Collect the culture supernatant and determine the bait protein concentration. If the bait protein concentration is high, dilute it to a final concentration of 500 μg/mL with 1×PBS for subsequent experiments.
(3) For pre-purified GST-tagged fusion protein:
Determine the bait protein concentration. If the bait protein concentration is high, dilute it to a final concentration of 500 μg/mL with 1×PBS for subsequent experiments.
Note: If the bait protein contains GSH, GSH must first be removed by dialysis or ultrafiltration before proceeding to subsequent steps.
4. Detection of Protein-Protein Interaction
(1) Immobilization of Bait Protein
1) Add the bait protein obtained in Step 3 to the pre-washed gel, mix gently, and incubate on a shaker at 4 °C for 3 h. Reserve a portion of the bait protein sample as an input control for later detection.
2) Separation by centrifugation. After incubation, centrifuge at 1250×g for 30 seconds at 4 °C, transfer the supernatant to a new centrifuge tube, and store for analysis.
3) Transfer the complex in the centrifuge tube to a spin column, centrifuge at 1250×g for 30 seconds at 4 °C, transfer the flow-through to a new centrifuge tube, and label as “bait flow-through” for later detection.
4) Add 500 μL of 1×PBS to the spin column, invert gently several times to wash the gel, centrifuge at 1250×g for 30 seconds at 4°C, and discard the eluate. Repeat 4 times to obtain the bait protein-gel complex.
(2) Capture of Target Protein.
1) Add 800 μL of the prepared prey protein sample to the bait protein-gel complex. Incubate with gentle shaking on a shaker at 4°C for at least 3 hours or overnight. Reserve a portion of the prey protein sample as a prey input for later detection.
Note: Longer incubation times may be required to ensure sufficient binding between bait and prey proteins, and can be adjusted according to experimental conditions.
2) After incubation, centrifuge at 1250×g for 30 seconds at 4 °C, transfer the flow-through to a new centrifuge tube, and label as “prey flow-through” for later detection.
3) Add 500 μL of 1×PBS to the spin column, invert gently several times to wash the gel, centrifuge at 1250×g for 30 seconds at 4 °C, and discard the eluate. Repeat 4 times to obtain the bait–prey protein–gel complex.
5. Elution of Bait–Prey Protein Complex: Elution can be performed using one of the following two methods, depending on the characteristics of the tagged protein and requirements of subsequent experiments.
(1) Non−Denaturing Elution.
Pipette 100 μL of the prepared GST elution buffer into the washed resin, mix the resin and elution buffer thoroughly, then centrifuge and collect the eluate. If elution is inefficient, increase the glutathione concentration or perform multiple elution.
(2) Elution with SDS−PAGE Loading Buffer.
For every 40 μL of original beads volume, add 50 μL of 1xPBS to resuspend the beads, followed by 50 μL of 2×SDS Loading Buffer. Mix gently by flicking the tube, then heat at 95-100°C for 5-10 minutes. Centrifuge at 1250×g for 30 seconds. Collect the supernatant for SDS-PAGE electrophoresis or Western Blot analysis.
Note: The recommended loading volume for WB is 20 μL or less. The remaining sample can be stored at -20°C.
I2621791
危险品化学品经营许可证（带存储）