Anti-HA 亲和层析介质以4%琼脂糖凝胶为基质，杂蛋白非特异性结合少。本试剂盒包含高质量的Anti-HA琼脂糖凝胶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HA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。
HA标签(HA-tag)、Myc标签(Myc-tag)、His标签(His-tag)、Flag标签(Flag-tag)、V5标签(V5-tag)和GST标签(GST-tag)等是表达载体上最常见的一些标签，通过与这些标签的融合表达可以非常方便地检测目的蛋白及与目的蛋白相互结合的蛋白，也可以非常方便地用于目的蛋白的纯化。
| A1455938A | Anti-HA 亲和层析介质
| 0.4 mL | 2 mL | 4°C | 每 250 µg 样品20µL
| | A1455938B | 1×裂解液 | 20 mL | 100 mL | 4°C | 每250 µg 样品150 µL | | A1455938C | HA多肽 | 0.2 mL | 1 mL | -20°C | 每100 µL 1×洗液10 µL | | A1455938D | 酸性洗脱液
| 2 mL | 10 mL | 4°C | 每 250 µg 样品100µL | | A1455938E | 10×洗液 | 40 mL | 200 mL | 4°C | 每 250 µg 样品 0.5 mL | | A1455938F | 中和液
| 0.2 mL | 1 mL | 4°C | 每 100 µL 酸性洗脱液10 µL | | A1455938G | 蛋白酶抑制剂(100x)
| 0.2 mL | 1 mL | -20°C | 每 150 µL 裂解液 1.5 µL | | A1455938H | 2×上样缓冲液 | 0.4 mL | 2 mL | -20°C | 每 250 µg 样品 20 µL
|
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. 凝胶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. Anti-HA resin 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。
使用说明：
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：HA 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HA tag Mouse mAb ( Ab106781 )、Recombinant HA tag Antibody ( Ab106771 )、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。
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) HA多肽洗脱液配置(1.5 mg/mL)：取一定量HA多肽储存液用洗液溶液进一步稀释HA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) 凝胶漂洗。轻轻重悬 Anti-HA Resin，尽量形成均匀的凝胶悬浊液，按照通常每 250 ug 中加入20μl混合均匀的凝胶悬浊液(以 下免疫沉淀步骤中都以每样加入20μl凝胶悬浊液为例)，取适量Anti-HA resin 至一洁净离心管中， 加入1x wash buffer 至最终体积为约0.5 mL。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) 加入凝胶与孵育。按照每250 μg 蛋白样品加入20 μL凝胶悬浊液的比例加入凝胶，置于侧摆摇床或旋转混合仪上，室温孵育2小时或4ºC孵育过夜。
b) 分离。孵育结束后，2000 rpm离心2min ，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。 2000 rpm离心2min，去除上清。重复使用 wash buffer 洗涤三次。得到蛋白-凝胶复合物。
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原始凝胶体积，加入100 μL HA多肽洗脱液，置于垂直旋转混合仪 4℃孵育 1-2 h，2000 rpm 离心 2 min，吸取上清液，收集洗脱组分，即为HA标签蛋白及其复合物，为了提高洗脱效率，可延长孵育时间或重复洗脱，HA标签蛋白及其复合物置于 4℃待用，-20℃或-80℃长期保存。如洗脱效果不佳，则可提高 HA 肽浓度或多次洗脱。
b) SDS-PAGE 上样缓冲液洗脱
每20ul原始凝胶体积加入30ul 的PBS（自备）重悬凝胶，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：SDS-PAGE 上样缓冲液洗脱会使目的蛋白能够完全从磁珠上脱离，但是抗体也会完全脱落。凝胶上 HA 抗体为鼠抗，后续 WB 实验建议选择兔抗。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原始凝胶体积，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1: 酸性洗脱法虽然高效，但仍可能低于竞争洗脱法或SDS-PAGE上样缓冲液洗脱法。
注2: 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HA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。
Anti-HA Affinity Chromatography Medium is based on a 4% agarose gel matrix, which exhibits low non-specific binding to miscellaneous proteins. This kit contains high-quality Anti-HA agarose gel and optimized, validated essential reagents for immunoprecipitation, making procedures such as immunoprecipitation (IP, also referred to as Pull-down) and co-immunoprecipitation (Co‑IP) simpler, more convenient, and highly efficient. It is widely applicable for the immunoprecipitation, co‑immunoprecipitation, or purification of HA-tagged fusion proteins and their associated protein complexes.
Tags like HA-tag, Myc-tag, His-tag, Flag-tag, V5-tag and GST-tag are among the most common tags found in expression vectors. The fusion expression of target proteins with these tags facilitates convenient detection of the target protein and proteins that interact with it, as well as simplifies the purification of the target protein.
| A1455938 | Components | 20T | 100T | Storage Temperature | Quantity Per Test | | A1455938A | Anti-HA resin | 0.4 mL | 2 mL | 4°C | 20 µL per 250 µg sample | | A1455938B | 1×Lysis Buffer | 20 mL | 100 mL | 4°C | 150 µL per 250 µg sample | | A1455938C | HA Peptide | 0.2 mL | 1 mL | -20°C | 1.5 µL per 100 µL wash Buffer | | A1455938D | Acid Elution Buffer | 3 mL | 15 mL | 4°C | 100 µL per 250 µg sample | | A1455938E | 10xWash Buffer | 40 mL | 200 mL | 4°C | 0.5 mL per 250 µg sample | | A1455938F | Neutralization Buffer | 0.3 mL | 1.5 mL | 4°C | 10 µL per 100 µL Acid Elution Buffer | | A1455938G | Protease Inhibitor Cocktail(100x) | 0.2 mL | 1 mL | -20°C | 1.5 µL per 150 µL Lysis buffer | | A1455938H | 2xSDS-PAGE Loading buffer | 0.6 mL | 3 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. Anti-HA resin usage should be optimized based on experimental requirements.
2. Before performing protein‑protein interaction operations, be sure to read this manual carefully.
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. The resin should be stored in the storage solution and protected from drying. Mix thoroughly before use.
5. Anti-HA resin 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 follow safety guidelines and adhere to laboratory reagent handling protocols.
8. The lysis buffer included in this kit already contains protease inhibitors. If special needs arise, other appropriate inhibitor cocktails may be considered.
9. Anti-HA resin must be fully resuspended before use, i.e., invert the tube several times to ensure thorough mixing.
10. After collection, protein samples should be purified as soon as possible and always kept at 4 °C or on ice to slow down protein degradation or denaturation.
Procedure (Unless otherwise specified, all procedures should be performed at 4°C)
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: HA 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: HA tag Mouse mAb ( Ab106781 )、Recombinant HA tag Antibody ( Ab106771 )、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
Buffering 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 HA Peptide Elution Buffer (10 mg/mL): Dilute a certain amount of the HA peptide stock solution with wash buffer to a final concentration of 150 μg/mL. For instance, add 15 μL of the peptide to 985 μL of wash buffer and mix well to achieve a final concentration of 150 μg/mL. Adjustments can be made according to experimental requirements.
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) Resin Washing: Gently resuspend the Anti-HA Resin 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 Anti-HA Resin into a clean centrifuge tube, and add 1x wash buffer to a final volume of approximately 0.5 mL. Repeat the above steps twice.
3. Preparation of Test Samples (Note: Perform all sample lysis steps at 4°C or on ice)
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）Add Resin and Incubate: Add the magnetic beads to the protein sample at a ratio of 20 µL bead suspension per 250 µg of protein sample. Place the tube on a rocking platform or rotator and incubate at room temperature for 2 hours or at 4°C overnight.
b) centrifugal separation: After incubation, Centrifuge at 2000 rpm for 2 minute at 4°C. Wait until all resin are captured, then transfer the supernatant to a new centrifuge tube. Retain this supernatant for potential future analysis.
c) Washing: Add 0.5 mL of Wash Buffer containing inhibitors and gently resuspend the beads by pipetting. Centrifuge at 2000 rpm for 2 minute at 4°C, then remove and discard the supernatant. Repeat this wash step three times using Lysis Buffer with inhibitors. This yields the bait protein-resin complex.
Note: The completeness of washing can also be assessed by measuring the OD280 of the wash supernatant. If the OD280 is greater than 0.05, appropriately increase the number of washes.
5. Protein Elution
Depending on the characteristics of the tagged protein and the requirements of subsequent experiments, choose one of the following three methods for elution.
a) Peptide Competitive Elution
Add 100 µL of HA Peptide Elution Buffer per 20 µL of original resin volume. Mix well and place the tube on a rocking platform or rotator. Incubate with shaking at room temperature for 30-60 minutes, or at 4°C for 1-2 hours. To improve elution efficiency, the incubation time can be extended or the elution step can be repeated. Gently mix 3-5 times during incubation. After incubation, Centrifuge at 2000 rpm for 2 minute at 4°C. Once the beads are fully captured, aspirate the supernatant into a new centrifuge tube for Western Blot analysis.
b) SDS-PAGE Loading Buffer Elution
Add 30 µL of PBS (self-prepared) per 20 µL of original bead volume to resuspend the beads. Then add 30 µL of 2xSDS Loading Buffer. Mix gently by shaking, then heat at 95-100°C for 5-10 minutes. Centrifuge at 2000 rpm for 2 minute a, and collect the supernatant for SDS-PAGE electrophoresis or Western Blot analysis.
Note: Elution with SDS-PAGE Loading Buffer enables complete detachment of the target protein from the beads, but the antibody will also be completely eluted. The Anti-HA antibody on the resin is mouse-derived; therefore, it is recommended to select a rabbit antibody for the subsequent WB experiment. The recommended loading volume for Western Blot detection is within 20 µL. The remaining sample can be stored frozen at -20°C.
c) Acid Elution
Add 100 µL of Acid Elution Buffer per 20 µL of original resin volume. Mix well and place the tube on a rocking platform or rotator. Incubate at room temperature for 5 minutes. Centrifuge at 1000 rpm for 1 minute at 4°C, then transfer the supernatant to a new centrifuge tube. If neutralization is required, add 10 µL of Neutralization Buffer to adjust the pH to neutral.
Note 1: Although efficient, acid elution may still be less effective than competitive elution or SDS-PAGE loading buffer elution.
Note 2: As differences in target proteins may influence the efficiency of acid elution, if high elution efficiency is required, the pH of the Acid Elution Buffer can be adjusted within the range of 2.5-3.0; correspondingly, the pH or volume of the Neutralization Buffer should also be adjusted. Specific experimental conditions need to be optimized by the user. Alternatively, consider using the potentially more efficient HA Peptide Competitive Elution method or the expected highest efficiency SDS-PAGE Loading Buffer Elution method. The disadvantage of the latter is that elution occurs under denaturing conditions, which may impact subsequent experiments requiring protein activity.
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