阿拉丁生产的生物素标记蛋白 Pull-Down 试剂盒（Magnetic Beads），专为研究生物分子相互作用设计，可高效捕获与生物素标记蛋白（诱饵蛋白）相互作用的靶蛋白（猎物蛋白），也可用于生物素标记蛋白复合物的 Pull-Down 与洗脱。
试剂盒内配套提供结合缓冲液、TBS 缓冲液、链霉亲和素磁珠、酸性洗脱液、中和缓冲液及变性洗脱用上样缓冲液，无需额外配制，操作简便。磁珠可高特异性结合生物素标记样品，搭配配套试剂，既能实现高效洗脱，又能保障实验效果稳定，结果可靠。本试剂盒可满足常规科研实验需求，助力蛋白相互作用、信号传导等相关研究高效开展。
使用说明：
1. 生物素标记反应和纯化：
a. 取适量待标记的已知蛋白，将其溶解于1X PBS中，终浓度0.2-2 mg/mL。若溶液中存在伯胺类物质（如Tris或Glycine）或者铵离子，为了确保后续实验的准确性和有效性，强烈推荐使用脱盐柱进行脱盐处理。
b. 生物的标记和纯化推荐参考阿拉丁生物素抗体标记试剂盒（Sulfo-NHS-LC-Biotin）(B1491943)的使用说明。
注：如果使用Avi标签蛋白生物素标记试剂盒(BirA法) (B1505408)，请按照该试剂盒的说明进行。
c. 生物素标记后的蛋白在去除游离生物素后可以直接使用，建议标记后蛋白浓度0.5-2mg/mL，如果浓度过高或者为冻干粉末状态可以用Binding Buffer进行稀释。
2. Streptavidin Magnetic Beads和生物素标记蛋白等样品的结合：
a. Streptavidin Magnetic Beads的准备：充分颠倒混匀Streptavidin Magnetic Beads，转移所需的体积到新的1.5ml离心管中，通常每个样品需加入40µl混合均匀的Streptavidin Magnetic Beads。
b. 加入Binding Buffer至最终体积约为0.5ml，用移液器轻轻吹打重悬Streptavidin Magnetic Beads。置于磁力架上分离30秒，去除上清(避免触及磁珠)，完成一次洗涤步骤。然后再按照前述洗涤步骤，洗涤1次，最终去除上清。
c. 样品结合：加入50-200µl生物素标记并纯化的样品，通常50µl Streptavidin Magnetic Beads的结合量为10-50µg蛋白，不宜超过50µg，颠倒混匀并翘板摇床摇动孵育2小时。
d. 孵育结束后，置于磁力架上分离1分钟，保留Streptavidin Magnetic Beads并收集上清。可通过测定上清中蛋白浓度或者进行SDS-PAGE分析来大致确定Streptavidin Magnetic Beads结合标记的已知蛋白样品的量。
e. 洗涤：使用500µl 1xTBS重悬Streptavidin Magnetic Beads，置于磁力架上分离1分钟，小心移去上清，吸头不要触到底部磁珠。重复本步骤1次。如果是对于生物素标记复合物的Pull down，可以直接进入到后续的洗脱步骤。
3. 目标蛋白等的Pull-Down：
a. 加入50-400µl样品裂解液(或其它含有目标蛋白等的样品)到Streptavidin Magnetic Beads中，轻轻吹打混合均匀。
注：选择合适的裂解液，用于制备细胞或组织的裂解液。优先推荐阿拉丁生产的RIPA裂解液(强) (R1506976)、RIPA裂解液(中) (R1505849)或RIPA裂解液(弱) (R1505850)用于样品的制备。如果使用自行配制的或其它公司生产的裂解液，需要确保裂解液的pH为6-8。细胞或组织裂解时，请注意添加适当的蛋白酶抑制剂以避免蛋白降解。
b. 4ºC翘板摇床摇动孵育至少2小时或者过夜，不可涡旋混合。
注：更高的结合量需要更长的孵育时间，每种蛋白的孵育时间需要自行摸索。如果目的蛋白比较稳定并且裂解液稳定，孵育也 可以在室温进行，但室温的孵育时间不宜过长。
c. 孵育结束后，置于磁力架上分离1分钟收集上清用于后续分析。吸取上清时，吸头不要触到底部磁珠。
d. 使用500µl 1xTBS重悬Streptavidin Magnetic Beads，置于磁力架上分离1分钟，小心收集上清，用于后续使用。吸头 不要触到底部磁珠。重复本步骤2次，即共洗涤3次。
4. 复合物的洗脱：
a. 变 性 洗 脱：
此方法洗脱的样品适用于SDS-PAGE检测。向洗涤后的复合物的Streptavidin Magnetic Beads中加入100 µL 1XSDS-PAGE 上样缓冲液混合均匀，95℃加热10 min。待冷却后，置于磁力架上分离1分钟。收集上清，进行SDS-PAGE检测。
b. 非变 性 洗 脱：
加入50µl的Elution Buffer重悬Streptavidin Magnetic Beads，摇床缓慢摇动洗脱5-10分钟，以洗脱所捕获的目标蛋白等。置于磁力架上分离1分钟，收集洗脱液，吸头不要触到底部磁珠。重复步骤5e-f 2次，即共洗脱3次，获得的洗脱液即为目标蛋白。第一次洗脱得到的复合物浓度最高，最后一次的最低，可根据实验需求取单次或混合后的洗脱样品进行后续的实验。洗脱液中立即加入Neutralization Buffer，推荐体积比例为Elution Buffer : Neutralization Buffer = 10:1。洗脱获得的样品置于4ºC待用，或者-20ºC长期保存。
注：：
1. Elution Buffer进行洗脱，就有可能发生链霉亲和素脱落，需注意孵育时间不要超过10分钟。
2. Elution Buffer能破坏绝大部分的抗体与抗原的相互作用。但为了确保更好的洗脱效果，可预先用300µl 0.1% Tween-20的水溶液洗涤磁珠1次。如果对洗脱效率的要求比较高，可用酸性洗脱液(8M Guanidine·HCl (pH1.5))进行洗脱，相应的中和液的pH值或量也要进行一定的调整，例如100μl酸性洗脱液(8M Guanidine·HCl (pH1.5))和15μl中和液(1M Tris-HCl(pH9.5)。
注意事项：
1. Streptavidin Magnetic Beads需维持pH为6-8，避免高速离心、干燥；请勿长时间将磁珠置于磁场中，否则可能会引起磁珠聚团。
2. Streptavidin Magnetic Beads使用前要适当充分重悬，即颠倒若干次使磁珠混合均匀，混匀操作须轻柔，不宜剧烈涡旋震荡等，避免蛋白变性。
3. 建议设置阴性和阳性对照组，以排除Streptavidin Magnetic Beads非特异性吸附的干扰。
4. 待结合分子的类型、大小及生物素标记方式和程度等都会影响结合效率，建议通过梯度稀释法来确定每种具体应用的磁珠用量，同时可以考虑加大磁珠用量至待结合分子2-3倍摩尔数量以确保结合充分。
The Biotinylated Protein Pull-Down Kit (Magnetic Beads) produced by Aladdin is specifically designed for studying biomolecular interactions. It enables efficient capture of target proteins (prey proteins) that interact with biotin-labeled proteins (bait proteins) and can also be used for the pull-down and elution of biotin-labeled protein complexes.
The kit includes binding buffer, TBS buffer, streptavidin magnetic beads, acid elution buffer, neutralization buffer, and denaturing loading buffer for elution, requiring no additional preparation and offering straightforward operation. The beads exhibit high specificity for binding biotin-labeled samples. When used with the provided reagents, they enable efficient elution while ensuring stable experimental performance and reliable results. This kit meets the needs of routine scientific research, facilitating efficient studies in protein-protein interactions, signal transduction, and related areas.
Instruction for use
1. Biotinylation Reaction and Purification:
a. Take an appropriate amount of the target protein to be labeled and dissolve it in 1X PBS to a final concentration of 0.2-2 mg/mL. If the solution contains primary amines (e.g., Tris or Glycine) or ammonium ions, it is highly recommended to perform desalting using a desalting column to ensure the accuracy and effectiveness of subsequent experiments.
b. For biotin labeling and purification, it is recommended to refer to the instructions of the Aladdin Biotin Antibody Labeling Kit (Sulfo-NHS-LC-Biotin) (Product No. B1491943).
Note: If using the Avi-tag Protein Biotinylation Kit (BirA Method) (Product No. B1505408), please follow the instructions provided with that kit.
c. After removal of free biotin, the biotin-labeled protein can be used directly. The recommended concentration for the labeled protein is 0.5-2 mg/mL. If the concentration is too high or the protein is in lyophilized powder form, it can be diluted with Binding Buffer.
2. Binding of Streptavidin Magnetic Beads to biotin-labeled protein and other samples.:
a. Preparation of Streptavidin Magnetic Beads: Thoroughly invert and mix the Streptavidin Magnetic Beads. Transfer the required volume to a new 1.5 ml centrifuge tube. Typically, add 40 µl of the well-mixed Streptavidin Magnetic Beads per sample.
b. Add Binding Buffer to a final volume of approximately 0.5 ml. Gently resuspend the Streptavidin Magnetic Beads by pipetting. Place the tube on a magnetic stand for 30 seconds, then carefully remove the supernatant (avoiding contact with the beads) to complete one wash step. Repeat the wash step once more as described, and finally remove the supernatant.
c. Sample Binding: Add 50–200 µl of the biotin-labeled and purified sample. Typically, 50 µl of Streptavidin Magnetic Beads can bind 10–50 µg of protein, and it is not recommended to exceed 50 µg. Invert to mix and incubate with rocking on a tilting shaker for 2 hours.
d. After incubation, place the tube on a magnetic stand for 1 minute. Retain the Streptavidin Magnetic Beads and collect the supernatant. The amount of known protein sample bound to the beads can be roughly determined by measuring the protein concentration in the supernatant or by SDS-PAGE analysis.
e. Washing: Resuspend the Streptavidin Magnetic Beads in 500 µl of 1× TBS. Place the tube on a magnetic stand for 1 minute, then carefully remove the supernatant, avoiding touching the beads at the bottom. Repeat this step once. For pull-down of biotin-labeled complexes, proceed directly to the subsequent elution step.
3. Pull-Down of Target Proteins:
a. Add 50–400 µl of sample lysate (or other samples containing the target protein) to the Streptavidin Magnetic Beads, and mix gently by pipetting.
Note: Select an appropriate lysis buffer for preparing cell or tissue lysates. It is recommended to use Aladdin's RIPA Lysis Buffer (Strong) (R1506976), RIPA Lysis Buffer (Medium) (R1505849), or RIPA Lysis Buffer (Weak) (R1505850) for sample preparation. If using self-prepared or other commercially available lysis buffers, ensure the pH is between 6 and 8. When lysing cells or tissues, be sure to add appropriate protease inhibitors to prevent protein degradation.
b. Incubate at 4°C on a rocking shaker for at least 2 hours or overnight. Do not vortex.
Note: Higher binding capacity may require longer incubation times, which should be optimized for each protein. If the target protein is stable and the lysis buffer is compatible, incubation can also be performed at room temperature, but the duration should not be excessively long.
c. After incubation, place the tube on a magnetic stand for 1 minute, and collect the supernatant for subsequent analysis. When aspirating the supernatant, avoid touching the beads at the bottom.
d. Resuspend the Streptavidin Magnetic Beads in 500 µl of 1× TBS, place the tube on a magnetic stand for 1 minute, and carefully collect the supernatant for later use. Avoid touching the beads at the bottom. Repeat this step twice, for a total of three washes.
4. Elution of Complexes:
a. Denaturing Elution:
This method is suitable for samples to be analyzed by SDS-PAGE. Add 100 µL of 1× SDS-PAGE loading buffer to the washed Streptavidin Magnetic Beads containing the complexes and mix thoroughly. Heat at 95 °C for 10 minutes. After cooling, place the tube on a magnetic stand for 1 minute. Collect the supernatant for SDS-PAGE analysis.
b. Native (Non‑Denaturing) Elution:
Resuspend the Streptavidin Magnetic Beads in 50 µL of Elution Buffer and gently shake on a rocking shaker for 5–10 minutes to elute the captured target proteins. Place the tube on a magnetic stand for 1 minute and collect the eluate, being careful not to touch the beads with the pipette tip. Repeat this elution step twice, for a total of three elutions. The collected eluates contain the target proteins. The first elution typically yields the highest concentration of complexes, while the last yields the lowest. Depending on experimental needs, either a single elution or a pooled sample may be used for downstream assays. Immediately add Neutralization Buffer to the eluate at a recommended volume ratio of Elution Buffer : Neutralization Buffer = 10 : 1. The eluted samples can be stored at 4 °C for short‑term use or at –20 °C for long‑term storage.
Notes:
When Elution Buffer is used, streptavidin detachment may occur; therefore, the incubation time should not exceed 10 minutes.
Elution Buffer disrupts most antibody‑antigen interactions. To ensure better elution efficiency, the beads may be pre‑washed once with 300 µL of 0.1 % Tween‑20 in water. If higher elution efficiency is required, acidic elution buffer (8 M Guanidine·HCl, pH 1.5) can be used. In this case, the pH or amount of the neutralization solution should be adjusted accordingly—for example, 100 µL of acidic elution buffer (8 M Guanidine·HCl, pH 1.5) may be neutralized with 15 µL of neutralization buffer (1 M Tris‑HCl, pH 9.5).
Matters needing attention
1. Streptavidin Magnetic Beads should be maintained at pH 6–8. Avoid high‑speed centrifugation and drying. Do not leave the beads in a magnetic field for extended periods, as this may cause bead aggregation.
2. Before use, thoroughly resuspend the Streptavidin Magnetic Beads by gently inverting the container several times to ensure uniform mixing. Handle gently; do not vortex or shake vigorously to prevent protein denaturation.
3. It is recommended to include negative and positive control groups to account for any nonspecific adsorption by the Streptavidin Magnetic Beads.
4. Binding efficiency can be influenced by factors such as the type and size of the molecule to be bound, as well as the method and extent of biotinylation. It is advisable to determine the optimal bead amount for each specific application using a gradient dilution approach. Additionally, consider using a 2 to 3 fold molar excess of beads relative to the target molecule to ensure sufficient binding.
J2609468
危险品化学品经营许可证（带存储）