阿拉丁Immunoprecipitation Kit (Protein A Agarose Gel) 是为免疫沉淀（IP）实验提供的经典琼脂糖凝胶法解决方案。
该技术利用特异性抗体识别并结合溶液中的目标蛋白（抗原），进而通过能高效结合抗体的介质，如本试剂盒提供的高质量重组Protein A琼脂糖凝胶，形成不溶性抗原-抗体-凝胶复合物。经离心分离，即可从复杂的样品混合物（如细胞裂解液）中特异性捕获并纯化出目的蛋白。
基于此原理，本试剂盒的沉淀产物适用于后续对目的蛋白或其相互作用的复合物组分进行多种分析，是Western Blot（蛋白免疫印迹）验证、质谱分析等下游检测技术的理想前处理工具。试剂盒提供的核心成分为经过优化的重组Protein A琼脂糖凝胶及必要的配套试剂，确保了实验过程的便捷、稳定与高效。
本试剂盒中的Protein A Agarose Gel (即 Protein A琼脂糖凝胶)可以特异性地结合相关抗体，并可以借助离心机非常便捷地应用于目的蛋白或其蛋白复合物的免疫沉淀或纯化等实验。其特点有：
(1)特异性强、靶蛋白结合量高。本试剂盒提供的Protein A Agarose Gel为50%的凝胶悬浊液，每毫升Protein A agarose Gel(沉淀物)可结合超过30mg human IgG，且特异性强，非特异性杂蛋白结合少。
(2)可重复使用多次，性价比高。在正常情况下，本产品用于相同抗体的纯化时可回收使用3-5次。如果用于免疫共沉淀检测蛋白与蛋白的相互作用，不推荐重复使用。
F1373494 | Components | 20T | 100T | Storage | Quantity Per Test |
F1373494A | IP Lysis Buffer | 50 mL | 250 mL | 2-8°C | 200μL |
F1373494B | TBS(10X) | 10 mL | 30 mL | 2-8°C | 50μL |
F1373494C | IP Protein A Agarose Gel | 0.4mL | 2mL | 2-8°C | 20μL per 500~1000 μg lysate |
F1373494D | Mouse IgG | 10μL | 50μL | -20°C | On Request |
F1373494E | Rabbit IgG | 10μL | 50μL | -20°C | On Request |
F1373494F | IP Elution Buffer | 2mL | 10mL | 2-8°C | 50μL |
F1373494G | IP Neutralization Buffer | 0.2mL | 1 mL | 2-8°C | 5μL |
F1373494H | SDS-PAGE Loading Buffer（2X） | 0.6mL | 3 mL | -20°C | 20μL |
注意事项：
1. 本试剂盒内包含的IP裂解液已包含蛋白抑制剂，如果有特殊需求，可考虑选择其它适当的抑制剂混合物。
2. 所有的样品裂解步骤宜在冰浴或4ºC操作，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. 本试剂盒提供的IP裂解液经反复测试，适合很多情况下的免疫沉淀时的样品裂解和后续的洗涤。但由于免疫沉淀蛋白样品的复杂性和特殊性，本裂解液不一定适合所有免疫沉淀样品的裂解与洗涤。在使用本试剂盒提供的裂解液和洗脱效果欠佳的情况下，需要自行对于裂解液和洗涤液进行摸索和调整。
4. Agarose Gel使用前一定要充分重悬，即充分颠倒若干次使混合均匀。
5. 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。
6. 使用此试剂盒时抗体将与免疫沉淀的抗原一起被洗脱。因此，在还原型 SDS-PAGE 凝胶或 Western blot 时至少产生三个蛋白条带：抗体重链（ 50 kDa）、轻链（ 25 kDa ）和抗原。如果抗体条带掩盖了免疫沉淀的抗原，则可用与免疫沉淀中不同种属的抗体做 Western blot 检测(如, 免疫沉淀用鼠抗的 IgG 而检测用兔抗的IgG)。 或者可以购买阿拉丁的构象特异性二抗（rp303272），可以直接避免抗体轻重链对于IP实验的干扰。
7. 本试剂盒提供的裂解液不仅用于样品裂解，也用于后续的洗涤步骤。
使用说明：
1. 样本处理：(注意：样品裂解需在4ºC或冰上操作)
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)；
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。
对于悬浮细胞的裂解和准备：
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。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。
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：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。
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20mg组织加入约100-200 μl 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：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。
2. 免疫复合物的制备：
a. 将 2- 10 μg亲和纯化的抗体（可用1XTBS进行稀释）与预处理的细胞裂解液在微量离心管中混合。每个IP反应总蛋白建议量是500 – 1000 μg，可使用BCA蛋白定量试剂盒测定。
b. 用 IP 裂解液稀释细胞裂解液混合物至300 – 600 μL。
c. 置于翻转混合仪上4℃孵育1小时至过夜，以形成免疫复合物。
注：样品用量和孵育时间取决于每个特定的抗体-抗原体系，并且可能需要优化实验方案以使产量最大化。以上方案是针对2-10 μg亲和纯化的抗体，且可以根据需要按比例放大。
3. Protein A琼脂糖凝胶预处理：
a. 轻轻重悬Protein A Agarose Gel，尽量形成均匀的凝胶悬浊液，取20μl凝胶悬浊液至一洁净离心管中加入预冷的1X TBS至最终体积为约0.5mL-1mL。
b. 轻轻重悬Protein A Agarose Gel，4ºC 翻转混匀5min。6000×g在4ºC离心60秒，小心去除上清，不要吸到凝胶。重复上述步骤两次 。
4. 捕获免疫复合物：
a. 结合：将抗原-抗体复合物 加入预处理后的Protein A Agarose Gel中，置于翻转混合仪上4℃孵育1~4 h。
b. 洗涤： 6000×g在4ºC离心60秒，小心去除上清，不要吸到凝胶。加入预冷的1X TBS或IP裂解液0.5mL-1mL。轻轻重悬Protein A Agarose Gel，4ºC 翻转混匀5min。6000×g在4ºC离心60秒，小心去除上清，不要吸到凝胶。重复上述步骤两次 。
c. 洗脱： 本试剂盒提供以下两种洗脱方案，操作者可根据后期检测的需要选择不同的洗脱方法。。变性洗脱适用于 Western blot 分析。非变性洗脱被中和后，适用于酶法或功能测定。
变性洗 脱：
此方法洗脱的样品适用于SDS-PAGE检测。向洗涤后的含抗原-抗体-复合物的Protein A Agarose Gel中加入20 µL 2XSDS-PAGE 上样缓冲液混合均匀，100℃加热10 min。待冷却后，6000×g，离心60秒。收集上清，进行SDS-PAGE检测。
非变性洗 脱：
此方法洗脱的样品保持原有的生物活性，可用于后期功能分析。向洗涤后的含抗原-抗体-复合物的Protein A Agarose Gel中加入50 µL 洗脱缓冲液 ，混匀后置于旋转混合仪上，室温孵育5分钟，孵育完毕后，6000×g 4ºC离心60秒，将上清转移到新的离心管中，并立刻加入5μL Neutralization Buffer ，适当混匀，将洗脱产物pH调节至中性，用于后期功能分析。
注：
a. 为了获得最大的洗脱效率，可重复该洗脱步骤若干次，并将相同样品合并。
b. 洗脱并中和的蛋白及其复合物置于4ºC待用或者-20ºC或-80ºC长期保存。
c. 酸性洗脱法虽然高效，但仍可能低于变性洗脱法。
d. 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，可对洗脱液的pH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多肽竞争洗脱法或效率预期最高的变性洗脱法，后者的缺点是在变性条件下进行洗脱，可能会对后续对于蛋白活性有要求的实验产生影响。
The Aladdin Immunoprecipitation Kit (Protein A Agarose Gel) provides a classic agarose bead-based solution for Immunoprecipitation (IP) experiments.
The technique utilizes a specific antibody to recognize and bind the target protein (antigen) in solution. This is followed by the use of a medium that efficiently binds antibodies—such as the high-quality recombinant Protein A Agarose Gel provided in this kit—to form an insoluble antigen-antibody-bead complex. Through centrifugation, the target protein can be specifically captured and purified from complex sample mixtures, such as cell lysates.
Based on this principle, the precipitated products obtained with this kit are suitable for subsequent analysis of the target protein or its interacting complex components. It serves as an ideal pre-processing tool for downstream detection techniques such as Western Blot verification and mass spectrometry analysis. The kit's core components include optimized recombinant Protein A Agarose Gel and necessary supporting reagents, ensuring a convenient, stable, and efficient experimental process.
The Protein A Agarose Gel in this kit can specifically bind to relevant antibodies and can be easily applied with a centrifuge for experiments such as immunoprecipitation or purification of the target protein or its protein complexes. Its features include:
(1) High specificity and high target protein binding capacity. The Protein A Agarose Gel provided in this kit is a 50% bead suspension. Each milliliter of settled Protein A Agarose Gel can bind over 30 mg of human IgG, exhibits high specificity, and has low non-specific protein binding.
(2) Reusable multiple times, cost-effective. Under normal conditions, this product can be recovered and reused 3-5 times for purification with the same antibody. Reuse is not recommended when used for co-immunoprecipitation to detect protein-protein interactions.
F1373494 | Components | 20T | 100T | Storage | Quantity Per Test |
F1373494A | IP Lysis Buffer | 50 mL | 250 mL | 2-8°C | 200μL |
F1373494B | TBS(10X) | 10 mL | 30 mL | 2-8°C | 50μL |
F1373494C | IP Protein A Agarose Gel | 0.4mL | 2mL | 2-8°C | 20μL pre 500~1000 μg lysate |
F1373494D | Mouse IgG | 10μL | 50μL | -20°C | On Request |
F1373494E | Rabbit IgG | 10μL | 50μL | -20°C | On Request |
F1373494F | IP Elution Buffer | 2mL | 10mL | 2-8°C | 50μL |
F1373494G | IP Neutralization Buffer | 0.2mL | 1 mL | 2-8°C | 5μL |
F1373494H | SDS-PAGE Loading Buffer（2X） | 0.6mL | 3 mL | -20°C | 20μL |
Precautions
1. The IP Lysis Buffer included in this kit contains protease inhibitors. If specific experimental requirements exist, consider adding other appropriate inhibitor cocktails.
2. All sample lysis procedures should be performed on ice or at 4°C to minimize potential protein degradation. If cell lysis is insufficient, it can be supplemented with sonication after adding the lysis buffer.
3. The IP Lysis Buffer provided in this kit has been extensively tested and is suitable for sample lysis and subsequent washing in many immunoprecipitation scenarios. However, due to the complexity and specificity of immunoprecipitated protein samples, this lysis buffer may not be optimal for all IP sample types. If suboptimal performance is observed with the provided lysis or wash buffers during experimentation, optimization of lysis and wash conditions should be performed by the user.
4. The Agarose Gel must be fully resuspended before use by inverting the vial several times to ensure a homogeneous mixture.
5. Protein samples should be purified as soon as possible after collection and must always be kept at 4°C or on ice to slow protein degradation or denaturation.
6. When using this kit, the antibody will be co-eluted with the immunoprecipitated antigen. Consequently, during reducing SDS-PAGE or Western blot analysis, at least three protein bands will be detected: the antibody heavy chain (approximately 50 kDa), light chain (approximately 25 kDa), and the antigen. If the antibody bands obscure the immunoprecipitated antigen, Western blot detection can be performed using an antibody from a different species than that used for immunoprecipitation (for example, using rabbit-derived IgG for detection if mouse-derived IgG was used for immunoprecipitation). Alternatively, Aladdin's conformation-specific secondary antibodies (rp303272) can be purchased to directly avoid interference from antibody heavy and light chains in IP experiments.
7. The lysis buffer provided in this kit is used for both sample lysis and the subsequent washing steps.
Instruction for use
1. Sample Preparation：(Note: Perform all sample lysis steps at 4°C or on ice)
* For 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.
* 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.
* 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.
* 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.
* 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.
2. Immune Complex Formation:
a. Combine 2-10 μg of affinity-purified antibody (diluted with 1X TBS) with the pre-cleared cell lysate in a microcentrifuge tube. The recommended total protein amount per IP reaction is 500–1000 μg, as determined by a BCA protein assay kit.
b. Dilute the cell lysate mixture with IP Lysis Buffer to a final volume of 300–600 μL.
c. Incubate on a rotator or rocking platform at 4°C for 1 hour to overnight to allow immune complex formation.
Note: The required sample amount and incubation time are dependent on the specific antibody-antigen system and may require optimization to maximize yield. The above protocol is designed for 2-10 μg of affinity-purified antibody and can be scaled up as needed.
3. Protein A Agarose Gel Preparation:
a. Gently resuspend the Protein A Agarose Gel to form a uniform suspension. Transfer 20 μl of the bead suspension to a clean microcentrifuge tube and add pre-chilled 1X TBS to a final volume of approximately 0.5-1 mL.
b. Gently resuspend the Protein A Agarose Gel and mix by inversion at 4°C for 5 minutes. Centrifuge at 6,000 × g for 60 seconds at 4°C. Carefully remove the supernatant without disturbing the bead pellet. Repeat this wash step twice.
4. Capture of Immune Complexes:
a. Binding: Add the antigen-antibody complex mixture to the prepared Protein A Agarose Gel. Incubate on a rotator or rocking platform at 4°C for 1-4 hours.
b. Washing: Centrifuge at 6,000 × g for 60 seconds at 4°C. Carefully aspirate the supernatant without disturbing the bead pellet. Add 0.5-1 mL of pre-chilled 1X TBS or IP Lysis Buffer. Gently resuspend the Protein A Agarose Gel and mix on a rotator at 4°C for 5 minutes. Centrifuge at 6,000 × g for 60 seconds at 4°C and carefully aspirate the supernatant. Repeat this wash procedure twice.
c. Elution: This kit provides two elution protocols. Users can select the appropriate method based on downstream application requirements. Denaturing elution is suitable for Western blot analysis. Non-denaturing elution, after neutralization, is suitable for enzymatic assays or functional studies.
Denaturing Elution:
This method is suitable for SDS-PAGE analysis. Add 20 µL of 2X SDS-PAGE Loading Buffer to the washed Protein A Agarose Gel containing the antigen-antibody complexes. Mix thoroughly. Heat at 100°C for 10 minutes. After cooling, centrifuge at 6,000 × g for 60 seconds. Collect the supernatant for SDS-PAGE analysis.
Non-denaturing Elution:
This method preserves the native biological activity of the eluted samples for subsequent functional analysis. Add 50 µL of Elution Buffer to the washed Protein A Agarose Gel containing the antigen-antibody complexes. Mix and incubate on a rotator at room temperature for 5 minutes. After incubation, centrifuge at 6,000 × g for 60 seconds at 4°C. Immediately transfer the supernatant to a new microcentrifuge tube and add 5 µL of Neutralization Buffer. Mix well to adjust the pH of the eluate to neutral for subsequent functional analysis.
Notes:
a. To maximize elution efficiency, repeat the elution step and pool the eluates.
b. Eluted and neutralized proteins/complexes can be stored at 4°C for immediate use or at -20°C/-80°C for long-term storage.
c. Although acidic elution is efficient, it may be less effective than denaturing elution.
d. Elution efficiency of the acidic method may vary depending on the target protein. If high elution efficiency is critical, optimize the pH of the elution buffer (with corresponding adjustments to the neutralization buffer). Alternatively, consider using potentially more efficient methods such as peptide competition elution or the highly efficient denaturing elution. Note that denaturing conditions may impact subsequent experiments requiring protein activity.
ZJ26F0130845
危险品化学品经营许可证（带存储）