His Pull down Kit是一款能够高效完成标签诱饵蛋白与靶蛋白共沉降的试剂盒。通过His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His固定于琼脂糖凝胶上，形成His-琼脂糖凝胶；将诱饵蛋白与His融合表达，获得的His-诱饵蛋白融合蛋白可与His-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-His-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His标签蛋白琼脂糖凝胶是将 IDA 配基通过 9 个原子的间隔臂以醚键固定在高刚性琼脂糖基质上，然后螯合上金属离子 Ni2+而制备成的一种介质。高刚性琼脂糖基质具有高流速、低反压的特点。高刚性琼脂糖基质具有更优的孔道结构，赋予了其高于传统琼脂糖基质的载量 。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. 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。
3. His标签蛋白纯化亲和层析介质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。
4. 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。
5. 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。
6. 请注意安全事项，遵守实验室试剂操作规范操作。
7. 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。
8. 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。
9. 在免疫沉淀或纯化时，建议设置阳性和阴性对照组。
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：His 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His tag (C-terminal) Mouse mAb ( Ab155835)、Recombinant His tag Antibody ( Ab086830)、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：准备1mL 250mM咪唑。即取62.5 µL 4M咪唑，加入到937.5 µL的1×洗液中。
(2) 1× Lysis Buffer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×洗液配制：按照 9:1 的比例用去离子水将 10×洗液稀释待用。例如：1mL 10×洗液加入 9 mL 去离子水，混匀后即为1×洗液。
(4) 凝胶漂洗。轻轻重悬His标签蛋白纯化亲和层析介质，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His标签蛋白纯化琼脂糖凝胶至一洁净离心管中，加入0.5 mL1×洗液，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. 诱饵蛋白准备
(1) 对于大肠杆菌表达的His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His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His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His，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°C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°C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°C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 µLHis标记的融合蛋白裂解液。对于已纯化好的His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) 向离心管中加入500 μL 1×PBS，倒置几次温和混匀，清洗凝胶，1250×g在4°C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°C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 μL 1×PBS，倒置几次温和混匀，清洗凝胶，1250×g在4°C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His洗脱液加入到清洗好后的填料中，充分混匀填料与洗脱液置于旋转平台上轻轻振荡，孵育 5-10 分钟，进行离心并收集洗脱液。
注：洗脱效果不佳，则可提高谷胱甘肽浓度或多次洗脱。
(2) 多肽竞争洗脱
每 40 µL原始凝胶体积，加入100 µL组氨酸肽洗脱缓冲液。将混合物置于垂直旋转仪上，孵育 5-10 分钟，随后1250×g在4°C离心30秒。吸取上清液，即为洗脱组分，其中含有组氨酸标签蛋白及其复合物。
注：若洗脱效果不佳，可提高组His多肽浓度或进行多轮洗脱。待凝胶珠完全沉降后，将上清液转移至新的离心管中，用于蛋白质免疫印迹（Western Blot）分析。
(3) SDS-PAGE 上样缓冲液洗脱
每40 µL原始凝胶体积加入50uL 的1×洗液重悬凝胶，再加入50 µL的2×SDS Loading Buffer，轻摇摇晃混匀后95-100°C高温处理5-10分钟。1250×g在4°C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 His Pull‑Down Kit is designed for efficient co‑sedimentation of tagged bait proteins with target proteins. It enables the isolation and purification of unknown captured pro-teins that interact with His‑tagged bait proteins. The simple experimental principle is as follows: His residues are immobilized on agarose beads to form His‑agarose resin; the bait protein is expressed as a fusion with a His‑tag, and the resulting His‑bait fusion protein binds to the His‑agarose resin. If a captured protein that interacts with the bait protein exists in the system, an agarose‑His‑bait protein‑captured protein complex will be formed, allowing the interacting captured protein to be isolated and detected.
This product utilizes high‑quality His‑tag protein agarose resin. This resin is prepared by immobilizing IDA ligands onto a highly rigid agarose matrix via an ether linkage with a 9‑atom spacer arm, followed by chelation with Ni²⁺ metal ions.The highly rigid agarose matrix supports high flow rates and low backpressure. Its optimized pore structure provides higher binding capacity compared with conventional agarose matrices. The kit features easy operation, strong binding capacity, and wide applicability.
Important Notes:
1. This product is intended for scientific research use by professionals only.
2. 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.
3. Ni-IDA Agarose 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.
4. Agarose Resin that has been boiled lose their binding capacity and should not be reused.
5. Reagent volumes can be adjusted according to experimental needs. It is recommended to perform preliminary experiments to validate immunoprecipitation conditions or optimize lysis parameters.
6. Please follow safety guidelines and adhere to laboratory reagent handling protocols.
7. The lysis buffer included in this kit already contains protease inhibitors. If specific needs arise, other appropriate inhibitor cocktails can be used.
8. 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.
9. It is recommended to include positive and negative control groups during immunoprecipitation or purification procedures.
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: His tag antibody.
b) Secondary Antibodies: Goat Anti-Mouse IgG H&L (HRP), Goat Anti-Rabbit IgG H&L (HRP).
c) Other Reagents: BST, 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: His tag (C-terminal) Mouse mAb (Ab155835), Recombinant His tag Antibody (Ab086830), 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) Preparation of Elution Buffer: Prepare 1 mL of 250 mM imidazole solution. Specifically, add 62.5 µL of 4 M imidazole to 937.5 µL of 1× Wash Buffer.
(2) 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 1: Lysis buffer containing inhibitors should be prepared fresh immediately before use and is not suitable for frozen storage and later use.
Note 2: This lysis buffer is suitable for mammalian cells.
(3) Preparation of 1×Wash Buffer: Dilute the 10×Wash Buffer with deionized water at a ratio of 9:1 before use. For example: add 9 mL of deionized water to 1 mL of 10× Wash Buffer, mix well to obtain 1×Wash Buffer.
(4) Ni-IDA Agarose Resin Washing: Gently resuspend the Ni-IDA Agarose 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 Ni-IDA agarose resin to a clean centrifuge tube. Add 0.5 mL of 1×wash buffer, 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 His-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 His-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 His-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.
4. Pull-Down Assay
(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 three methods, depending on the characteristics of the tagged protein and requirements of subsequent experiments.
(1) Non-Denaturing Elution
Pipette 100 μL of the prepared 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.
Note: If elution efficiency is unsatisfactory, the glutathione concentration may be increased or multiple elution steps may be performed.
(2) Peptide competitive elution
Add 100 µL of His‑peptide elution buffer to every 40 µL of the original gel volume. Incubate the mixture on a vertical rotator for 5-10 minutes, then centrifuge at 1250×g and 4 °C for 30 seconds. Aspirate the supernatant to obtain the eluted fraction, which contains the His‑tagged protein and its complexes.
Note: If elution efficiency is unsatisfactory, the His‑peptide concentration can be increased or multiple rounds of elution can be performed. Once the gel beads are fully settled, transfer the supernatant to a new centrifuge tube for Western Blot analysis.
(3) Elution with SDS-PAGE Loading Buffer
For every 40 μL of original beads volume, add 50 μL of 1×PBS 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.
ZJ26F0535413
危险品化学品经营许可证（带存储）