背景介绍：
UltraBio™ Anti-Flag Magnetic Beads，即Anti-Flag磁珠，也称Anti- DYKDDDDK Magnetic Beads、Anti-Flag免疫磁珠或Flag抗体磁珠， Flag纳米抗体磁珠偶联了经过严格筛选、优化并重组表达的无轻、重链的Flag纳米抗体可用于捕获哺乳动物、植物、细菌、酵母、昆虫等多种生物细胞、组织提取物中的Flag标签（ DYKDDDDK ）融合蛋白及其相互作用蛋白。本试剂盒包含高质量的Anti-Flag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)或免疫共沉淀(Co-IP)实验更加简单、便捷、高效，广泛用于带有Flag标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。
His标签(His-tag)、Myc标签(Myc-tag)、Flag标签(Flag-tag)、HA标签(HA-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。
注意事项：
1.本试剂盒内包含的IP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。
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.Magnetic Beads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.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100-200μL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. Anti-Flag Magnetic Beads预处理：
a. 轻轻重悬Anti-Flag Magnetic Beads，尽量形成均匀的悬浊液，取20μl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.置于磁力架上分离1min。小心去除上清，不要吸到磁珠。重复上述步骤两次 。
3. 样品的结合：
a. 将细胞裂解液与预处理过的Anti-Flag Magnetic Beads在离心管中混合。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. 置于翻转混合仪上4℃孵育4-6 h或者过夜（也可选择常温1-2h）。
c. 将上述混合液置于磁力架上静置1min，然后把上清液转移到新的离心管中备用(上清液可用于检测DYKDDDDK标签蛋白是否存在残留)，原离心管中剩余的即为蛋白-磁珠复合物。
d. 向所得的蛋白-磁珠复合物中加入1mL 1XTBS缓冲液，用移液器轻柔吹打重悬，接着在磁力架上静置1min后，吸弃上清，重复上述步骤两次 。
4. 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。
a.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Anti-Flag Magnetic Beads中加入20 µL 1XSDS-PAGE 上样缓冲液（可用5X上样缓冲液进行稀释）混合均匀，100℃加热10 min。待冷却后，置于磁力架上分离1min。收集上清，进行SDS-PAGE检测。
b.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Anti-Flag Magnetic Beads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，置于磁力架上分离1min，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。
Background Introduction:
UltraBio™ Anti-Flag Magnetic Beads, also known as Anti-Flag magnetic beads, Anti-DYKDDDDK Magnetic Beads, Anti-Flag immunomagnetic beads, or Flag antibody magnetic beads, are conjugated with rigorously screened, optimized, and recombinantly expressed heavy/light chain-free Flag nanobodies. These beads are designed to capture Flag-tagged (DYKDDDDK) fusion proteins and their interacting proteins from cell or tissue extracts of various biological sources, including mammals, plants, bacteria, yeast, and insects.This kit includes high-quality Anti-Flag magnetic beads and optimized, validated essential reagents for immunoprecipitation, making Immunoprecipitation (IP) or Co-Immunoprecipitation (Co-IP) experiments simpler, more convenient, and efficient. It is widely used for the immunoprecipitation, co-immunoprecipitation, or purification of Flag-tagged fusion proteins or their protein complexes.
Tags such as His-tag, Myc-tag, Flag-tag, HA-tag, V5-tag, and GST-tag are among the most commonly used epitope tags in expression vectors. Fusion expression with these tags facilitates convenient detection of the target protein and its interacting partners, as well as efficient purification of the protein of interest.
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 Magnetic Beads 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.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. Anti-Flag Magnetic Beads Preparation:
a. Gently resuspend the Anti-Flag Magnetic Beads 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. Place the tube on a magnetic rack for 1 min to separate the beads, then carefully remove and discard the supernatant without disturbing the beads. Repeat this wash procedure two additional times.
3. Sample Binding
a. Combine the cell lysate with the pre-treated Anti-Flag Magnetic Beads in a microcentrifuge tube. The recommended total protein input per IP reaction is 500–1000 μg, as determined by a BCA protein assay kit.
b. Incubate the mixture on a rotator or rocking platform at 4°C for 4–6 hours or overnight (alternatively, incubation at room temperature for 1–2 hours is also acceptable).
c. Place the tube on a magnetic rack and let it stand for 1 minute. Carefully transfer the supernatant to a new microcentrifuge tube (the supernatant can be used to check for any residual DYKDDDDK-tagged protein). The remaining material in the original tube is the protein-bead complex.
d. Add 1 mL of 1X TBS buffer to the obtained protein-bead complex. Gently resuspend the beads by pipetting. Place the tube on the magnetic rack for 1 minute, then carefully aspirate and discard the supernatant. Repeat this wash procedure two additional times.
4. Elution:
This kit provides two elution protocols. The operator can choose the appropriate method based on downstream application requirements. Denaturing elution is suitable for Western blot analysis, while non-denaturing elution—after neutralization—is applicable for enzymatic assays or functional studies.
a. Denaturing Elution:
Samples eluted using this method are suitable for SDS-PAGE analysis. Add 20 µL of 1X SDS-PAGE loading buffer (prepared by diluting 5X loading buffer, if applicable) to the washed Anti-Flag Magnetic Beads containing the antigen-antibody complexes. Mix thoroughly and heat at 100°C for 10 minutes. After cooling, place the tube on a magnetic rack for 1 minute. Collect the supernatant for SDS-PAGE analysis.
b. 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 Anti-Flag Magnetic Beads containing the antigen-antibody complexes. Mix and incubate on a rotator at room temperature for 5 minutes. After incubation, place the tube on a magnetic rack for 1 minute. Transfer the supernatant to a new microcentrifuge tube and immediately 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.
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