1. 本产品仅限于专业人员的科学研究使用。
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°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。
4. Anti-V5纳米抗体磁珠填料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。
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. 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。
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. 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。
10. 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：V5 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V5 tag Mouse mAb (Ab133823)、Recombinant V5 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) 1×裂解液配制：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1ml的裂解液中加入10 μL 蛋白酶抑制剂(100×)，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：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。
(2) 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。
(3) 磁珠漂洗。轻轻重悬Anti-V5 纳米抗体磁珠，尽量形成均匀的磁珠悬浊液，按照通常每 250 µg 中加入20 μL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20 μL磁珠悬浊液为例)至一洁净离心管中，加入1×洗液至最终体积为约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)吸取磁珠悬浊液会比较方便。
3. 待测样品准备
(1) 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)
(2) 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100-200 μL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2-10分钟。充分裂解后，用细胞刮刀刮落细胞，收集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。
(3) 悬浮细胞样品裂解
250-1000×g室温离心5 分钟收集细胞；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100-200 μL的比例加入含抑制剂裂解液。混匀后置于冰上孵育5-20分钟 (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。
(4) 对于细菌或酵母样品的裂解和准备：
对于1 mL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100-200 μL含抑制剂裂解液，轻轻vortex或者弹击管底以混匀，冰上裂解2-10分钟。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。
(5) 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 12000×g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。
4. 免疫沉淀(Immunoprecipitation, IP)
(1) 加入磁珠与孵育。
按照每250 μg 蛋白样品加入20 μL磁珠悬浊液的比例加入磁珠，置于侧摆摇床或旋转混合仪上，室温孵育2 h或4ºC孵育过夜。
(2) 磁力架静置分离。
孵育结束后，在磁力架上静置1分钟，将上清转移至新的离心管中，留样备检。
(3) 洗涤。
加入0.5 mL的洗液，用移液器轻轻吹打重悬磁珠。在磁力架上静置1分钟，去除上清。重复使用洗液洗涤三次。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，进行洗脱。
SDS-PAGE 上样缓冲液洗脱: 每20 µL原始磁珠体积加入30 µL的洗液重悬磁珠，再加入30 µL的2×上样缓冲液，轻摇摇晃混匀后95-100℃高温处理5-10分钟。5000×g 离心20秒，取上清进行SDS−PAGE 电泳或进行Western 检测。
注1: Anti-V5 纳米抗体磁珠没有抗体轻重链污染问题，建议首选变性洗脱法，洗脱效率更高。
注 2：Western Blot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。
Important Notes
1. This product is for research use only and restricted to scientific research by qualified professionals.
2. Please read this instruction manual carefully before performing protein–protein interaction assays.
3. Unless otherwise stated, all procedures are recommended to be performed at 4°C to minimize protein degradation. If cell lysis is incomplete, sonication may be applied after treatment with lysis buffer.
4. Anti-V5 Nanobody magnetic beads should be stored in storage solution to prevent drying. Mix thoroughly by gentle inversion several times before use. Vigorous vortexing is not recommended to avoid antibody denaturation.
5. Boiled beads lose their binding capacity and should not be reused.
6. Reagent volumes may be adjusted according to actual experimental conditions. Preliminary experiments are recommended to verify immunoprecipitation or optimize lysis conditions.
7. Please observe safety precautions and comply with standard laboratory reagent handling protocols.
8. The lysis buffer included in this kit already contains protease inhibitors. Other appropriate inhibitor cocktails may be used for specific requirements.
9. Protein samples should be purified promptly after collection and kept at 4°C or on ice to reduce protein degradation or denaturation.
10. Positive and negative controls are recommended for immunoprecipitation or purification experiments.
Procedure (Unless otherwise specified, all procedures should be per-formed at 4°C)
1 Reagent Preparation
Additional Required Materials:
(1) Reagents to be prepared by the user:
a) Primary Antibody: V5 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: V5 tag Mouse mAb (Ab133823), Recombinant V5 tag Antibody (Ab086830), Goat Anti-Rabbit IgG H&L (HRP)(Ab176443), Goat Anti-Mouse IgG H&L (HRP)(Ab179001) and Goat Anti-Rabbit IgG H&L (HRP)(Ab170144).
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) 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: The inhibitor-containing lysis buffer should be prepared fresh before use and should not be frozen and stored for later applications.
(2) reparation of 10×Wash Buffer: Dilute the 10×wash buffer with deionized water at a 9:1 ratio. For example, add 9 mL of deionized water to 1 mL of 10×wash buffer, and mix well to obtain the 1×wash buffer.
(3) Beads Washing: Gently resuspend the Anti-V5 Magnetic 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 Anti-V5 Magnetic Beads into a clean centrifuge tube, and add 1×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)
(1) 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.
(2) 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.
(3) 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 number of cells, 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.
(4) 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.
(5) 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)
(1) 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.
(2) 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.
(3) 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
Elution with SDS-PAGE Loading Buffer: For every 20 μL of original bead volume, add 30 μL of wash to resuspend the beads, followed by 30 μL of 2×SDS Loading Buffer. Mix gently by flicking the tube, then heat at 95-100°C for 5-10 minutes. Centrifuge at 5000×g for 1 minute. Collect the supernatant for SDS-PAGE electrophoresis or Western Blot analysis.
Note 1: Since Anti-V5 nanobody magnetic beads are free from antibody heavy/light chain contamination, the denaturing elution method is preferred for higher elution efficiency.
Note 2: The recommended loading volume for WB is 20 μL or less. The remaining sample can be stored at -20°C.
ZJ26F0737312
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