His标签免疫沉淀试剂盒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。 试剂盒包含高质量的His-tag琼脂糖磁珠及经过优化验证的免疫沉淀必要试剂，使免疫沉淀(Immunoprecipitation, IP，也称Pull-down)或免疫共沉淀(Co-IP)实验更加简单、便捷、高效，广泛用于带有His标签的融合蛋白或其蛋白复合物的免疫沉淀、免疫共沉淀或纯化等实验。 。
本产品由高品质的His标签蛋白琼脂糖磁珠 （IDA-Ni）可以用于各种表达来源（如大肠杆菌、酵母、昆虫细胞和哺乳动物细胞）的组氨酸标签（6xHis-tagged）蛋白的纯化。它是以磁性琼脂糖磁珠微球为基础，通过化学方法偶联了三配位的亚氨基二乙酸（IDA），螯合镍离子（Ni²⁺）后，可以形成比较稳定的骨架结构，从而有更多的位点与组氨酸标签上的咪唑环继续配位，达到结合目的蛋白的效果。具备操作简便、结合能力强、适用范围广等优点。具备操作简便、结合能力强、适用范围广等优点。
本试剂盒提供的 Ni IDA Magarose Beads 为50%凝胶悬浊液，包装体积为总体积，每毫升中含有0.5ml纯凝胶(沉淀物)。对于常规的免疫沉淀实验，按照每 250μg 样品使用20μl凝胶悬液。
| H1506147 | Components | 10T | 40T | Storage Temperature | Quantity Per Test | | H1506147A | His标签蛋白纯化琼脂糖磁珠 (IDA-Ni)
| 0.2 mL | 0.8 mL | 4°C | 20 µL per 250 μg sample | | H1506147B | 1× 裂解液
| 10 mL | 40 mL | 4°C | 150 µL per 250 μg sample | | H1506147C | His 多肽 | 0.1 mL | 0.4 ml | -20°C | 10 µL per 100 μL wash Buffe | | H1506147D | 酸性洗脱液
| 1mL | 4 mL | 4°C | 100 µL per 250 μg sample | | H1506147E | 10 × 洗液
| 20 mL | 80 mL | 4°C | 0.5 mL per 250 μg sample | | H1506147F | 中和液
| 0.1 mL | 0.4 mL | 4°C | 10 µL per 100 μL Acid Elution Buffer | | H1506147G | 蛋白酶抑制剂(100×)
| 0.1 mL | 0.4 mL | -20°C | 1.5 µL per 150 μL Lysis buffer | | H1506147H | 2 ×上样缓冲液
| 0.2 mL | 0.8 mL | -20°C | 20 µL per 250 μg sample |
注: 对于常规的免疫沉淀实验，按照每250 μg样品使用20μl的层析介质。 |
注意事项
1. Ni IDA Magarose Beads用量可按实验实际进行取用。
2. 在进行蛋白互作操作之前，请务必认真阅读此说明书。
3. 除非另有说明，所有操作建议于4℃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. 填料应保存在储存溶液中，防止干燥，使用前请充分混匀。
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. 本产品仅限于专业人员的科学研究使用。
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. Ni IDA Magarose Beads使用前一定要充分重悬，即充分颠倒若干次使混合均匀。
10. 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。
使用方法
1. 试剂准备
下列可能用到的试剂及材料未提供，需额外准备：
(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
(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)、 Recombinant Protein A/G, HRP conjugate(rp303272)、Goat Anti-Mouse IgG H&L (HRP) (Ab179001)
2. 溶液配置
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。(除非另有说明，所有操作建议于4℃下进行)
a) 按照每50-100万细胞使用100-200μl含抑制剂裂解液用于裂解，配制适量的含抑制剂裂解液。将Lysis Buffer与Protease Inhibitor Cocktail (100x)按照100:1的 比例混合，例如在1ml的Lysis Buffer中加入10μl Protease Inhibitor Cocktail (100x)，即得1ml含抑制剂裂解液(Lysis Buffer with Protease Inhibitor Cocktail)。配制好的含抑制剂裂解液宜放置在冰浴或4ºC。
注：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。
b) His多肽洗脱液配置(1.5 mg/mL)：取一定量His多肽储存液用洗液 溶液进一步稀释His多肽溶液至150 μg/mL。即 10 μL的多肽，加入到 90 μL 的wash buffer中，混匀后终浓度为150 μg/mL。也可根据实验需求进行调整
c) 1x wash buffer 配置：按照 9:1 的比例用去离子水将 10 x wash buffer 稀释待用。例如：1mL 10xwash buffer 加 入 9mL 去离子水，混匀后即为 1× wash buffer。
d) 磁珠漂洗。轻轻重悬Ni IDA Magarose Beads ，尽量形成均匀的凝胶悬浊液，按照通常每 250 ug 中加入20μl混合均匀的磁珠悬浊液(以下免疫沉淀步骤中都以每样加入20μl凝胶悬浊液为例)至一洁净离心管中， 加入1x wash buffer 至最终体积为约0.5mL。重复上述步骤两次。
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. 待测样品准备
a) 对于血清样本的准备:
若目标蛋白丰度较高，建议用裂解液稀释血清样本至目标蛋白终浓度为 50~150 µg/mL，置于冰上备用(或置于-20℃长期保存)
b) 对于贴壁细胞的裂解和准备：
吸除培养液，用PBS洗涤2次，然后吸净残留的液体。按照每50-100万细胞(相当于6孔板的一个孔)加入100-200μl的含抑制剂裂解液，适当吹打，使裂解液和细胞充分接触。通常裂解液接触动物细胞1-2秒后，细胞就会被裂解。植物细胞宜在冰上裂解2-10min。充分裂解后，用细胞刮刀刮落细胞，收集至 1.5 mL 离心管内10,000-14,000×g在4ºC离心3-5分钟，取上清，进行蛋白浓度测定后即可进行后续的免疫沉淀或免疫共沉淀等。注：裂解后会出现少量不溶性物质，主要为基因组DNA等，离心后会产生沉淀物。
c) 悬浮细胞样品裂解
250-1000×g室温离心5min收集细胞；用PBS洗涤2次，然后吸净残留的液体。轻轻vortex或者弹击管底以把细胞尽量分散开。按照每50-100万细胞加入100-200μl的比例加入含抑制剂裂解液。混匀后置于冰上孵育5~20 min(期间混匀几次)，轻弹管底或适当吹打，以充分裂解细胞向细胞团块中加入预冷 Lysis Buffer（含抑制剂按1%加），每50-100万细胞加入150-250 μL的比例加入含抑制剂裂解液；充分裂解后应没有明显的细胞沉淀。如果细胞量较多，建议分装成50-100万细胞/管，然后再裂解。大团的细胞较难裂解充分，而少量的细胞由于裂解液容易和细胞充分接触，相对比较容易裂解充分。充分裂解后，10,000-14,000×g在4ºC离心3-5分钟，取上清，进行蛋白浓度测定后即可进行后续的免疫沉淀和免疫共沉淀等。
注：裂解后会出现少量不溶性物质，主要为基因组DNA等，离心后会产生沉淀物。
d) 对于细菌或酵母样品的裂解和准备：
对于1ml菌液或酵母液，离心去上清，用PBS洗涤2次，然后吸净残留的液体。轻轻vortex或者弹击管底以把细菌或酵母尽量分散开。加入100-200μl含抑制剂裂解液，轻轻vortex或者弹击管底以混匀，冰上裂解2-10min。如果希望获得更好的裂解效果，细菌和酵母可以分别使用溶菌酶(lysozyme)和破壁酶(lyticase)消化，然后再使用含抑制剂裂解液进行裂解。充分裂解后，10,000-14,000×g在4ºC离心3-5分钟，取上清，进行蛋白浓度测定后即可进行后续的免疫沉淀和免疫共沉淀等。
注1：裂解后很可能会出现少量不溶性物质，主要为基因组DNA等，离心后会产生沉淀物。
注2：如果是可溶蛋白，直接取上清进行使用，如果是包涵体，则取沉淀，对包涵 体进行洗涤以及变性复性。
e) 对于组织样品的裂解和准备：
把组织剪切成细小的碎片，按照每20毫克组织加入约100-200微升裂解液的比例加入裂解液，然后用玻璃匀浆器或其它匀浆设备进行匀浆。充分匀浆可以确保组织被完全裂解。裂解后4ºC 12000g离心5分钟，取上清，进行蛋白浓度测定后即可进行后续的免疫沉淀和免疫共沉淀等。
注1：裂解后很可能会出现少量不溶性物质，主要为基因组DNA等，离心后会产生沉淀物。
注2：对于所有样品如果样本中目标蛋白丰度较低，或结合物间的结合较弱，可以增加初始样本量，同时等比例增加裂解缓冲液和酶抑制剂的用量，但总孵育体积最大不超过离心管体积的2/3，体积过大可以更换大规格离心管。
4. 免疫沉淀(Immunoprecipitation, IP)
a) 加入磁珠与孵育。按照每250 μg 蛋白样品加入20 μL磁珠悬浊液的加入新的微量离心管中，置于磁力架上静置1分钟，室温孵育2小时或4ºC孵育过夜。
b) 分离。孵育结束后，置于磁力架上静置1分钟 ，将上清转移至新的离心管中，留样备检。
c) 洗涤。加入0.5 mL的wash buffer，用移液器轻轻吹打重悬凝胶。置于磁力架上静置1分钟 ，去除上清。重复使用 wash buffer 洗涤三次。得到蛋白-磁珠复合物。
注：也可以通过检测洗涤得到的液体的OD280来判断是否洗涤完全，若OD280大于0.05，应适当增加洗涤次数。
5. 蛋白洗脱
根据标签蛋白的特点及后续实验要求，可以选择如下3种方法之一进行洗脱。
a) 非变性洗脱法
每20 μL原始磁珠体积，加入100 μL His多肽洗脱液，置于垂直旋转混合仪 4℃孵育 1-2 h，置于磁力架上静置1分钟 ，吸取上清液，收集洗脱组分，即为His蛋白及其复合物，为了提高洗脱效率，可延长孵育时间或重复洗脱，His标签及其复合物置于 4℃待用，-20℃或-80℃长期保存。如洗脱效果不佳，则可提高His多肽浓度或多次洗脱。
b) SDS-PAGE 上样缓冲液洗脱
每20ul原始磁珠体积加入30ul 的PBS（自备）重悬磁珠，再加入30uL的2×SDS Loading Buffer，轻摇摇晃混匀后95-100℃高温处理5-10分钟。1000 rpm 离心1min，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 20uL 以内，剩余的样本可冻存于-20℃保存。
c) 酸性洗脱
每20 μL原始磁珠体积，加入100 μL Acid Elution Buffer (酸性洗脱液)，混匀后置于侧摆摇床或旋转混合仪上，室温孵育5分钟。1000 rpm 离心1 分钟，将上清转移到新的离心管中。如需中和中性缓冲液，可加入10 μL Neutralization Buffer (中和液)，调整pH至中性。
注: 由于目的蛋白的差异可能对酸性洗脱法的洗脱效率有一定的影响，如果对洗脱效率的要求比较高，可对酸性洗脱 液的pH在2.5-3.0之间进行一定的调整，相应的中和液的pH值或量也要进行一定的调整，具体需要自行优化相关实验条件。 也可以考虑采用效率可能更高的His多肽竞争洗脱法或效率预期最高的SDS-PAGE上样缓冲液洗脱法，后者的缺点是在 变性条件下进行洗脱，可能会对后续的对于蛋白活性有要求的实验产生影响。
The His-tag Immunoprecipitation Kit is designed for efficient co-precipitation of tagged bait proteins and target proteins. The kit contains high-quality His-tag agarose magnetic beads along with optimized and validated essential reagents for immunoprecipitation, making Immunoprecipitation (IP, also known as Pull-down) or Co-Immunoprecipitation (Co-IP) experiments simpler, more convenient, and more efficient. It is widely used for immunoprecipitation, co-immunoprecipitation, or purification of His-tagged fusion proteins or their protein complexes.
This product contains high-quality His-tagged protein agarose magnetic beads (IDA-Ni), suitable for purifying histidine-tagged (6xHis-tagged) proteins from various expression sources, such as E. coli, yeast, insect cells, and mammalian cells. Based on magnetic agarose bead microspheres, the beads are chemically coupled with a tridentate iminodiacetic acid (IDA) ligand. After chelation with nickel ions (Ni²⁺), a relatively stable backbone structure is formed, providing more sites for coordination with the imidazole rings on the histidine tag, thereby effectively binding the target protein. The kit offers advantages including simple operation, strong binding capacity, and broad applicability.
The Ni IDA Magarose Beads provided in this kit is a 50% gel suspension, with the packaging volume referring to the total volume. Each milliliter contains 0.5 ml of pure gel (precipitate). For routine immunoprecipitation experiments, use 20 μl of gel suspension per 250 μg of sample.
| H1506147 | Components | 10T | 40T | Storage Temperature | Quantity Per Test | | H1506147A | Ni IDA Magarose Beads | 0.25 mL | 0.8 mL | 4°C | 20 µL per 250 μg sample | | H1506147B | 1 × Lysis Buffer | 10 mL | 40 mL | 4°C | 150 µL per 250 μg sample | | H1506147C | His Peptide
| 0.1 mL | 0.4 mL | -20 °C | 1.5 µL per 100 μL wash Buffer | | H1506147D | Acid Elution Buffer | 1 mL | 4 mL | 4°C | 100 µL per 250 μL sample | | H1506147E | 10x Wash Buffer
| 20 mL | 80 mL | 4°C | 500 µL per 250 μg sample | | H1506147F | Neutralization Buffer
| 0.1 mL | 0.4 mL | 4°C | 10 µL per 100 μL Acid Elution Buffer
| | H1506147G | Protease Inhibitor Cocktail(100 × ) | 0.1 mL | 0.4 mL | -20 °C | 1.5 µL per 150 μL Lysis buffer | | H1506147H | 2 × SDS-PAGE Loading buffer | 0.25 mL | 0.8 mL | -20 °C | 20 µL per 250 μg sample |
Note: For conventional immunoprecipitation experiments, use 20 μl of chromatography media per 250 μg of sample. |
Precautions
1. The amount of Ni IDA Magarose Beads used can be adjusted according to the actual experimental needs.
2. Before performing protein - protein interaction experiments, please read this manual carefully.
3. Unless otherwise specified, all procedures are recommended to be carried out at 4°C to minimize potential protein degradation. If cell lysis is incomplete, sonication may be used after applying the lysis buffer.
4. The beads should be stored in storage solution to prevent drying. Mix thoroughly before use.
5. Beads that have been boiled lose their binding capacity and should not be reused.
6. This product is intended for research use by qualified professionals only.
7. Please observe safety precautions and follow laboratory reagent handling protocols.
8. The lysis buffer included in this kit already contains protease inhibitors. If specific requirements exist, other appropriate inhibitor cocktails may be considered.
9. Before use, Ni IDA Magarose Beads must be fully resuspended by inverting several times to ensure a homogeneous mixture.
10. After collection, protein samples should be purified as soon as possible and kept at 4°C or on ice at all times to slow down protein degradation or denaturation.
Procedure
1. Reagent Preparation
The following reagents and materials that may be required for the experiment are not provided and need to be prepared separately:
(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: TBST, Electrophoresis Buffer, Transfer Buffer, Reducing SDS-PAGE Gel
(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)、 Recombinant Protein A/G, HRP conjugate(rp303272)、Goat Anti-Mouse IgG H&L (HRP) (Ab179001)
2. Solution Preparation
uffering agents provided in the kit can be used, or alternatively, different buffer systems can be prepared according to actual requirements. It is recommended that all buffers be filtered through a 0.22 μm or 0.45 μm membrane prior to use. Buffers should be stored at 4°C. Discard immediately if the reagent appears turbid.
a) 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 per 0.5–1 million cells. Mix Lysis Buffer and Protease Inhibitor Cocktail (100x) at a ratio of 100:1. For example, add 10 μl of Protease Inhibitor Cocktail (100x) 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.
b) Preparation of His Peptide Elution Buffer (10 mg/mL): Dilute a certain amount of the His peptide stock solution with wash buffer to a final concentration of 150 μg/mL. For instance, add 1.5 μL of the peptide to 98.5 μL of wash buffer and mix well to achieve a final concentration of 150 μg/mL. Adjustments can be made.
c) Preparation of 10x Wash Buffer: Dilute the 10x wash buffer with deionized water at a 9:1 ratio. For example, add 9 mL of deionized water to 1 mL of 10x wash buffer, and mix well to obtain the 1x wash buffer.
d) Beads Washing: Gently resuspend the Ni IDA Magarose Beads to form a homogeneous gel suspension. Typically, use 20 μl of the well-mixed gel suspension per 250 μg (the following immunoprecipitation steps are described based on adding 20 μl of gel suspension per sample). Transfer an appropriate amount of Ni IDA Magarose Beads into a clean centrifuge tube, and add 1x wash buffer to a final volume of approximately 0.5 mL. Allow to stand on the magnetic rack for 1 minute and discard the supernatant. Repeat the above steps twice.
Note: Using wide-bore tips (e.g., by cutting off the tip end with scissors) may facilitate pipetting of the gel suspension.
3. Preparation of Test Samples (Note: Perform all sample lysis steps at 4°C or on ice)
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
a) For the preparation of serum samples:
If the target protein is abundant, dilute the serum sample with Lysis Buffer to a final target protein concentration of 50–150 µg/mL. Keep the diluted sample on ice for immediate use or store at -20°C for long-term preservation.
b) For Adherent Cell Lysis and Preparation:
Aspirate the culture medium and wash the cells twice with PBS. Remove all residual liquid completely. Add 100-200 µL of Lysis Buffer with inhibitors per 0.5-1 million cells (equivalent to one well of a 6-well plate). Pipette gently to ensure thorough contact between the lysis buffer and cells. Animal cells are typically lysed within 1-2 seconds of contact with the buffer. For plant cells, lyse on ice for 2-10 minutes. After complete lysis, use a cell scraper to detach the cells and transfer the lysate to a 1.5 mL microcentrifuge tube. Centrifuge at 10,000-14,000 × g for 3-5 minutes at 4°C. Collect the supernatant for protein concentration determination before proceeding to subsequent immunoprecipitation or co-immunoprecipitation experiments.
Note: A small amount of insoluble material, primarily genomic DNA, may be present after lysis and will form a pellet upon centrifugation.
c) For Suspension Cell Lysis and Preparation:
Collect cells by centrifugation at 250-1,000 × g for 5 minutes at room temperature. Wash the pellet twice with PBS and remove all residual liquid completely. Gently vortex or tap the tube to disperse the cells. Add 100-200 µL of Lysis Buffer with inhibitors per 0.5-1 million cells. Mix and incubate on ice for 5-20 minutes (mix several times during incubation). Tap the tube or pipette gently to ensure complete cell lysis; no significant cell pellet should remain after thorough lysis. If processing a large number of cells, it is recommended to aliquot them into tubes containing 0.5-1 million cells per tube before lysis. Large cell clumps are more difficult to lyse completely, whereas smaller numbers of cells allow better contact with the lysis buffer and lyse more efficiently. After complete lysis, centrifuge at 10,000-14,000 × g for 3-5 minutes at 4°C. Collect the supernatant for protein concentration determination before subsequent immunoprecipitation or co-immunoprecipitation experiments.
Note: A small amount of insoluble material, primarily genomic DNA, may be present after lysis and will form a pellet upon centrifugation.
d) For Bacterial or Yeast Sample Lysis and Preparation:
For 1 mL of bacterial or yeast culture, centrifuge to pellet the cells and remove the supernatant. Wash the pellet twice with PBS and remove all residual liquid completely. Gently vortex or tap the tube to resuspend and disperse the bacterial or yeast cells. Add 100-200 µL of Lysis Buffer with inhibitors. Gently vortex or tap the tube to mix, and lyse on ice for 2-10 minutes. For improved lysis efficiency, bacteria and yeast can be pretreated with lysozyme and lyticase, respectively, before adding the Lysis Buffer with inhibitors. After complete lysis, centrifuge at 10,000-14,000 × g for 3-5 minutes at 4°C. Collect the supernatant for protein concentration determination before subsequent immunoprecipitation or co-immunoprecipitation experiments.
Note: A small amount of insoluble material, primarily genomic DNA, is likely present after lysis and will form a pellet upon centrifugation.
e) For Tissue Sample Lysis and Preparation:
Mince the tissue into small fragments. Add approximately 100-200 µL of Lysis Buffer per 20 mg of tissue. Homogenize the mixture using a glass homogenizer or other suitable homogenization device. Thorough homogenization ensures complete tissue lysis. After lysis, centrifuge at 12,000 × g for 5 minutes at 4°C. Collect the supernatant for protein concentration determination before subsequent immunoprecipitation or co-immunoprecipitation experiments.
Note: A small amount of insoluble material, primarily genomic DNA, is likely present after lysis and will form a pellet upon centrifugation.
4. Immunoprecipitation (IP)
a) Beads Addition and Incubation. Add magnetic beads to the protein sample at a ratio of 20 μL bead suspension per 250 μg of protein. Incubate the mixture on a rocking platform or rotary mixer for 2 hours at room temperature or overnight at 4°C.
b) Separation Using a Magnetic Rack. After incubation, place the tube on a magnetic rack and let it stand for 1 minute. Transfer the supernatant to a new microcentrifuge tube and save it for potential future analysis.
c) Washing. Add 0.5 mL of Wash Buffer and resuspend the beads by gently pipetting up and down. Place the tube on the magnetic rack and let it stand for 1 minute. Discard the supernatant. Repeat the washing process three times using Wash Buffer. The protein-beads complexes are now obtained.
Note: The completeness of washing can also be monitored by measuring the OD280 of the wash solution. If the OD280 reading is greater than 0.05, increase the number of washes appropriately.
5. Protein Elution
Baseds on the characteristics of the tagged protein and the requirements of downstream experiments, choose one of the following three elution methods.
a) Non-Denaturing Elution
For every 20 μL of the original bead volume, add 100 μL of His‑peptide elution buffer. Incubate the mixture on a vertical rotator at 4 °C for 1–2 h, then place it on a magnetic stand for 1 minute. Aspirate the supernatant to collect the eluted fraction, which contains the His‑tagged protein and its complexes. To improve elution efficiency, the incubation time may be extended or elution may be repeated. The His‑tagged protein and its complexes can be stored at 4 °C for immediate use, or at –20 °C/–80 °C for long‑term storage. If elution is insufficient, the His‑peptide concentration can be increased or multiple elution steps can be performed. Once the beads are fully captured, aspirate the supernatant into a new centrifuge tube for Western Blot analysis.
b) Elution with SDS-PAGE Loading Buffer
For every 20 μL of original bead volume, add 30 μL of PBS (user to provide) to resuspend the beads, followed by 30 μL of 2xSDS Loading Buffer. Mix gently by flicking the tube, then heat at 95-100°C for 5-10 minutes. Centrifuge at 1000 rpm for 1 minute. 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.
c) Acidic Elution
For every 20 μL of original bead volume, add 100 μL of Acid Elution Buffer. Mix thoroughly and incubate on a rocking platform or rotary mixer for 5 minutes at room temperature. Centrifuge at 1000 rpm for 1 minute and transfer the supernatant to a new microcentrifuge tube. If neutralization is required, add 10 μL of Neutralization Buffer to adjust the pH to neutral.
Note: The efficiency of acidic elution may vary depending on the specific target protein. If high elution efficiency is critical, the pH of the Acid Elution Buffer can be optimized within the range of pH 2.5-3.1. Accordingly, the pH or volume of the Neutralization Buffer should also be adjusted. Specific conditions need to be optimized by the use.
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