Protein A/G Magnetic Beads （蛋白质A/G磁珠）

Protein A/G Magnetic Beads 为 IP、Co-IP 和 ChIP 实验提供了一种快速便捷的方法。

描述和优势

MCE蛋白A/G磁珠通常用于从血清、细胞培养上清液或腹水中分离抗体，并用于从细胞或组织提取物中免疫沉淀和共免疫沉淀抗原。蛋白A/G磁珠包含重组蛋白A/G，其结合蛋白A和蛋白G的IgG结合域。

在免疫沉淀过程中，只需要少量的磁珠，非特异性结合很低。
•方便、省时。
•低非特异性结合。
•最小样本损失。
•抗体结合能力高达0.5-0.8 mg/mL。
•稳定的单瓶溶液。

Publications

• •Immunity. 2021 Sep 15;S1074-7613(21)00361-7.
• •Immunity. 2021 Apr 13;54(4):632-647.e9.
• •Cell metab. 2022 Jun 7;S1550-4131(22)00191-7.
• •Cell Mol Immunol. 2022 May 30;1-15.
• •Neuron. 2021 Mar 17;109(6):957-970.e8.
• •Signal Transduct Target Ther. 2022 Feb 28;7(1):54.
• •Signal Transduct Target Ther. 2021 Apr 16;6(1):152.
• •ACS Nano. 2021 Sep 3.
• •Nat Commun. 2022 Aug 9;13(1):4680.
• •Nat Commun. 2022 Mar 10;13(1):1248.
• •Nat Commun. 2022 Jan 19;13(1):386.
• •Nat Commun. 2021 May 14;12(1):2809.
• •Nat Commun. 2019 Nov 8;10(1):5091.
• •Nat Commun. 2019 Sep 25;10(1):4353. 
• •Adv Sci (Weinh). 2021 Feb 18;8(9):2004635.
• •Adv Sci (Weinh). 2021 Feb 8;8(8):2002874.

Storage

Stored at 4°C, and is stable for up to 2 years.

Do not centrifuge, dry or freeze the magnetic beads.

Protocol

1.   Preparation of Magnetic Beads

1.1   Resuspend the Magnetic Beads in the vial (tilt and rotate for 2 minutes or gently pipette for 10 times).

1.2   Transfer 25-50 μL of Protein A/G Magnetic Beads into a 1.5 mL tube (Transfer amount may be adjusted as required).

1.3   Add 400 μL of binding/wash buffer to the beads and gently pipette to mix. Place the tube into a magnetic stand to collect the beads against the side of the tube (Hereinafter referred to as magnetic separation). Remove and discard the supernatant. Repeat this step for 2 times.

2.   Binding of Antibody

2.1   Dilute antibody (Ab) to the final concentration of 5-50 μg/mL with binding/wash buffer. The optimal amount of Ab may be adjusted as required.

2.2   Add 400 μL of diluted Ab to the Protein A/G Magnetic Beads. Rotate tube for 30 minutes at room temperature or 2 hours at 4°C.

2.3   Perform magnetic separation. Transfer the supernatant into a new tube for further analysis, if desired. The supernatant is the non-binding fraction.

2.4   Add 400 μL of binding/wash buffer to the beads and gently pipette to mix. Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant. Repeat this step for 4 times.

3.   Immunoprecipitation of Target Antigen

3.1   Remove the tubes from the magnetic separator and add your sample containing the antigen (Ag) (typically 5-50 μg in 400 μL binding/wash buffer) and gently pipette to resuspend the Protein A/G Magnetic Beads-Ab complex.

3.2   Incubate with rotation for 30 minutes at room temperature or 2 hours at 4°C to allow Ag to bind to the Protein A/G Magnetic Beads-Ab complex.

3.3   Perform magnetic separation. Remove and discard the supernatant.

3.4   Wash the Magbeads-Ab-Ag complex 5 times using 400 μL binding/wash buffer for each wash. Perform magnetic separation between each wash, remove supernatant and resuspend by gentle pipetting.

3.5   Resuspend the Protein A/G Magnetic Beads-Ab-Ag complex in 400 μL binding/wash buffer and transfer the bead suspension into a clean tube. This is recommended to avoid co-elution of the proteins bound to the tube wall.

4.   Elution

This is a non-denaturation elution method.

4.1   Perform magnetic separation and remove the supernatant. Add 400 μL of binding/wash buffer into the tube and rotate for 5 minutes. Perform magnetic separation for 1 minute and remove the supernatant. Then add 25-50 μL elution buffer into the tube with magnetic beads-Ab-Ag complex, rotate for 5 minutes.

4.2   Perform magnetic separation, collect the supernatant.

4.3   The final solution can be used as samples for denaturing SDS-PAGE. Or the elution can be adjusted to neutral pH with neutralization buffer immediately and used for further analysis.