IP/Co-IP Kit (Protein A/G Magnetic Beads)

1. Buffer Preparation:

Notes:

a. After thawing, it is recommended to aliquot the IP lysis buffer appropriately to minimize repeated freeze-thaw cycles.

b. Prepared TBS-T should be stored at 4°C and can be kept for at least one month.

c. Reducing or non-reducing protein loading buffer may be used according to the sample requirements.

2. Antigen Sample Preparation:

Note: For cell lysis in a 6-well plate, 150-250 μL of lysis buffer may also be added to each well.

3. Magnetic Bead Pretreatment:

Thoroughly resuspend the magnetic beads. Transfer 25 μL of magnetic beads into a 1.5 mL EP tube, add 200 μL of binding/wash buffer, mix thoroughly, place the tube on a magnetic stand for magnetic separation, and discard the supernatant. Repeat the washing procedure twice.

Note: To ensure an even distribution of the magnetic beads, mix them thoroughly by repeated inversion, gentle vortexing, or using a mixer.

4. Immunocomplex Preparation:

The required sample amount and incubation time depend on the specific antibody-antigen system and may need to be optimized to achieve the maximum yield. The following protocol is designed for 2-20 μg of affinity-purified antibody and may be scaled proportionally as needed.

1. In a centrifuge tube, combine the cell lysate from each sample with 2-10 μg of immunoprecipitation antibody.

The protein concentration can be measured using a Protein Concentration Assay Kit (MCE Cat. No.: HY-K2001). A total protein amount of 500-1000 μg is recommended for each immunoprecipitation reaction.

2. Dilute the antigen-antibody reaction mixture to 500 μL with binding/wash buffer, mix thoroughly, and incubate on a rotator at room temperature for 1-2 h or at 4°C overnight to form the immunocomplex.

5. Immunocomplex Precipitation:

1. Add the antigen-antibody immunocomplex prepared above to the pretreated magnetic beads and incubate on a rotator at room temperature for 1-2 h or at 4°C for 2-4 h. Perform magnetic separation and collect the magnetic beads. Transfer the supernatant to a new EP tube for subsequent analysis.

2. Add 500 μL of binding/wash buffer to the centrifuge tube, mix gently, and perform magnetic separation. Collect the magnetic beads and transfer the supernatant to a new EP tube for subsequent analysis. Repeat the washing procedure twice.

6. Antigen Elution:

Two elution methods are provided. Select the appropriate method according to the experimental requirements.

1. Acidic Elution: Samples eluted using this method retain their original biological activity and can be used for subsequent functional analysis.

Add 100 μL of elution buffer to the centrifuge tube, mix thoroughly, and incubate at room temperature for 5-10 min. Perform magnetic separation, collect the supernatant, and immediately add neutralization buffer at 1/10 of the total eluate volume to neutralize the pH. The sample can then be used for subsequent functional analysis.

Note: The eluted protein can be stored at 4°C for a short period. For long-term storage, it is recommended to store the sample at -20°C.

2. Denaturing Elution: Samples eluted using this method are suitable for SDS-PAGE analysis.

Add 100 μL of protein loading buffer (1×) to the centrifuge tube, mix thoroughly, and heat at 95°C for 5 min. Perform magnetic separation, collect the supernatant, and analyze it by SDS-PAGE using the prestained protein marker supplied with the kit (10-190 kDa).

Notes:

a. Dilute the protein loading buffer to 1× with electrophoresis running buffer or ultrapure water. Reducing or non-reducing protein loading buffer may be selected according to the electrophoresis conditions.

b. At least three protein bands should be present in reducing SDS-PAGE or Western blot results: the antibody heavy chain (50 kDa), antibody light chain (25 kDa), and the antigen.