基本描述

产品名称

Protein A/G磁珠

别名

英文别名

Protein A/G MagBeads | Immunomagnetic Beads Protein A/G | Protein A/G Magnetic Beads

规格或纯度

BioReagent, for IP, for CoIP, 10 mg/mL, 200nm

产品介绍

Protein A/G磁珠是粒径为 200nm的纳米磁珠表面上共价结合了大量的 Protein A/G 蛋白，纳米级磁珠提供的超大比表面积，具有更多的结合位点，磁珠使用量更少，非特异性吸附率低。重组 Protein A/G 蛋白包含Protein A的5个免疫球蛋白结合区域和Protein G 的2个结合区域，结合能力较单一的 Protein A 和 Protein G 有很大提高。本产品广泛应用于细胞裂解物、细胞分泌上清、血清、腹水等样品中抗原的免疫沉淀（IP）或免疫共沉淀（Co-IP）。由于采用磁性分离，使得每次 IP 和 Co-IP操作流程可以节省 40%的时间。

产品特性

基质	硅基磁珠
配体	重组 Protein A/G 蛋白
粒径	200nm
磁珠浓度	10mg/mL
结合能力	≥0.7mg hIgG/mL 磁珠
适用范围	IP, Co-IP, ChIP, RIP 等
保质期	在2~8℃稳定保存两年

操作流程

贴壁细胞样品：

1. 移去培养基，用 PBS 洗细胞两次。

2. 收集细胞至 1.5mL EP 管内，按比例加入 IP Lysis/Wash Buffer，同时加入 PMSF 等相应的抑制剂，混匀后置于冰上静置 5-20min（期间混匀几次）。 3. 4℃, 12000-16000xg,10min 离心收集上清液，置于冰上以备后续实验（或置于-80℃长期保存）。

悬浮细胞样品：

1. 4℃、500-1000xg、10min，收集细胞，弃上清。

2. 用 PBS 洗细胞一次，即用 PBS 将细胞团重悬，4℃、500-1000xg、5min，收集细胞，弃上清。

3. 用预冷的 IP Lysis/Wash Buffer 重悬细胞。每 50mg细胞使用 500μL IP Lysis/Wash Buffer。同时加入 PMSF

等相应的抑制剂，混匀后置于冰上静置 5-20min（期间混匀几次）。

4. 4℃,12000-16000xg,10min 离心收集上清液，置于冰上以备后续实验（或置于-80℃长期保存）。

血清样品：

一般建议建议用 IP Lysis/Wash Buffer 稀释血清样品至目标蛋白终浓度为 50~150µg/mL，置于冰上备用（或置于-20℃长期保存）。

免疫复合物的制备

注意：样品所需的量和孵育时间均依赖于每个特定的抗体-抗原体系，因而可能需要优化才能得到最大产量。

以下实验方案针对 2-10μg 亲和纯化的抗体，根据需要可以按比例放大。

1. 在离心管中，将每个样品的细胞裂解液与 2-10μg 免疫沉淀抗体结合。每个免疫沉淀反应推荐的总蛋白量为 500-1500μg。

2. 用IP Lysis/Wash Buffer将抗体以及制备好的样品稀释至 300-500μL。

3. 在室温下孵育 1-2h，或 4℃ 2-4h，以形成免疫复合物。

免疫沉淀：

注意：为保证磁珠均匀分布，使用前通过反复颠倒或轻微涡旋混匀瓶中磁珠。

1. 将 20-50µL 的 UltraBio™ Protein A/G 磁珠加入1.5mL 离心管中。

2. 向磁珠中加入 500µL 预冷 PBS，轻柔混匀。

3. 将离心管放入磁力架中收集磁珠到离心管的一边。去除上清。

4. 向离心管中加入 200-500µL IP Lysis/Wash Buffer。颠倒离心管数次或轻微涡旋混匀 1min。用磁力架收集磁珠。去除上清。

5. 将抗原样品/抗体混合物加入装有磁珠的离心管中，保持混匀室温下孵育 1-2h，或 4℃ 2-4h。

6. 用磁力架收集磁珠，除去未结合的样品，保存以备分析。

7. 向离心管中加入 1000µL IP Lysis/Wash Buffer，轻柔混匀磁珠 5-10min。收集磁珠，弃上清。再重复洗两次。

8. 变性洗脱：向离心管中加入 80-100µL SDS-PAGE Sample Loading Buffer（1×），将样品置于 100℃水浴或者金属浴中加热 10min。通过磁力架分离磁珠，保留含有目的抗原的上清。

备注:如需保持蛋白活性，也可采用以下洗脱方式。

低 pH 洗脱：向离心管中加入 100µL Elution Buffer。保持混匀在室温下孵育离心管 5-10min。通过磁力分离磁珠，保留含有目的抗原的上清。每 100µL 洗出液中加入20µL Neutralization Buffer 来中和低 pH。

注意事项

1. 进行实验操作之前，请务必认真阅读本操作说明书。

2. 请勿高速离心、干燥或冷冻磁珠，这些操作会导致磁珠聚集而降低结合能力。

3. IP 实验中不同类型的抗体与抗原结合的亲和性是有区别的，抗体与抗原结合还会受到 IP Lysis/Wash Buffer 的影响，因此可自行优化操作细节或者筛选及配制缓冲液进行实验。

4. 磁珠使用前应充分振荡均匀。磁珠应保存在储存溶中，防止干燥。

5. 本产品仅供科学研究使用。

UltraBio™ Protein A+G Magnetic Beads are 200 nm nanoscale magnetic beads with a large amount of Protein A/G covalently bound to their surface. The nanoscale size of the beads provides an extremely high surface area, resulting in more binding sites. This means less magnetic bead usage is required, and non-specific binding is minimized. The recombinant Protein A/G contains five immunoglobulin-binding domains from Protein A and two binding domains from Protein G, significantly enhancing binding capacity compared to single Protein A or Protein G. This product is widely used for immunoprecipitation (IP) or co-immunoprecipitation (Co-IP) of antigens from cell lysates, cell culture supernatants, serum, ascites, and other samples. The magnetic separation feature allows each IP and Co-IP procedure to save up to 40% of the time.

Sample Preparation

Adherent Cell Samples:

1. Remove the culture medium and wash the cells twice with PBS.

2. Collect the cells into a 1.5 mL EP tube, add IP Lysis/Wash Buffer in proportion, and include inhibitors such as PMSF. Mix well and place on ice for 5-20 minutes (mixing several times during this period).

3. Centrifuge at 4°C, 12000-16000xg, for 10 minutes to collect the supernatant. Keep the supernatant on ice for subsequent experiments (or store at -80°C for long-term storage).

Suspension Cell Samples:

1. Centrifuge the cells at 4°C, 500-1000xg, for 10 minutes, and discard the supernatant.

2. Wash the cells once with PBS by resuspending the cell pellet in PBS, then centrifuge again at 4°C, 500-1000xg, for 5 minutes, and discard the supernatant.

3. Resuspend the cells in pre-cooled IP Lysis/Wash Buffer. Use 500 µL of IP Lysis/Wash Buffer for every 50 mg of cells. Add inhibitors such as PMSF, mix well, and place on ice for 5-20 minutes (mixing several times during this period).

4. Centrifuge at 4°C, 12000-16000xg, for 10 minutes to collect the supernatant. Keep the supernatant on ice for subsequent experiments (or store at -80°C for long-term storage).

Serum Samples:

1. It is generally recommended to dilute serum samples with IP Lysis/Wash Buffer to achieve a final protein concentration of 50-150 µg/mL. Keep the diluted samples on ice (or store at -20°C for long-term storage).

Preparation of Immune Complexes

Note: The amount of sample required and the incubation time depend on the specific antibody-antigen system and may need optimization to achieve maximum yield. The following protocol is designed for 2-10 µg of affinity-purified antibody and can be scaled up as needed.

1. In a centrifuge tube, combine the cell lysate with 2-10 µg of immunoprecipitation antibody for each sample. The recommended total protein amount for each immunoprecipitation reaction is 500-1500 µg.

2. Dilute the antibody and prepared sample to 300-500 µL with IP Lysis/Wash Buffer.

3. Incubate at room temperature for 1-2 hours, or at 4°C for 2-4 hours to form immune complexes.

Immunoprecipitation

Note: To ensure even distribution of the magnetic beads, mix the beads thoroughly by repeated inversion or gentle vortexing before use.

1. Add 20-50 µL of UltraBio™ Protein A+G Magnetic Beads to a 1.5 mL centrifuge tube.

2. Add 500 µL of pre-cooled PBS to the beads and mix gently.

3. Place the centrifuge tube on a magnetic rack to collect the beads on one side of the tube. Remove the supernatant.

Add 200-500 µL of IP Lysis/Wash Buffer to the tube. Mix by inverting the tube several times or gentle vortexing for 1 minute. Collect the beads using the magnetic rack and remove the supernatant.

4. Add the antigen sample/antibody mixture to the tube containing the beads. Keep the mixture homogenous and incubate at room temperature for 1-2 hours, or at 4°C for 2-4 hours.

5. Collect the beads using the magnetic rack, remove the unbound sample, and save it for analysis.

6. Add 1000 µL of IP Lysis/Wash Buffer to the tube, mix the beads gently for 5-10 minutes. Collect the beads and discard the supernatant. Repeat the wash twice more.

7. Denaturing Elution: Add 80-100 µL of SDS-PAGE Sample Loading Buffer (1×) to the tube. Heat the sample in a water bath or metal bath at 100°C for 10 minutes. Separate the beads using the magnetic rack and retain the supernatant containing the target antigen.

Note: If protein activity needs to be maintained, the following elution method can be used.

Low pH Elution: Add 100 µL of Elution Buffer to the tube. Mix well and incubate at room temperature for 5-10 minutes. Separate the beads using the magnetic rack and retain the supernatant containing the target antigen. Neutralize the low pH by adding 20 µL of Neutralization Buffer to every 100 µL of eluate.

Precautions

1. Please read the instruction manual carefully before performing the experiment.

2. Do not centrifuge at high speed, dry, or freeze the magnetic beads, as these operations can cause bead aggregation and reduce binding capacity.

3. Different types of antibodies have varying affinities for antigens in IP experiments, and the binding can also be affected by the IP Lysis/Wash Buffer. Therefore, you may optimize the operational details or select and formulate the buffer for your experiment.

4. Mix the magnetic beads thoroughly before use. Store the beads in their storage solution to prevent drying.

5. This product is for research use only.

储存与运输

物理形态	液体
浓度	10 mg/mL, 200nm
储存温度	2-8°C储存
运输条件	冰袋运输
稳定性与储存	Store at 2-8℃ (24 months). Upon receipt, it is recommended to aliquot.