Immunoprecipitation (IP) Detailed Protocol (For Reference Only)

The core principle of immunoprecipitation (IP) relies on the specific binding between a target protein and a specific antibody, followed by the capture of the antibody-antigen complex using a medium (e.g., Protein A/G). This enables the isolation and purification of the target protein from complex samples. The detailed procedures are as follows:

I. Pre-Experimental Preparation

Reagent Preparation:

Basic Reagents: Cell lysis buffer (e.g., RIPA lysis buffer, supplemented with protease inhibitors and phosphatase inhibitors to prevent protein degradation/dephosphorylation), PBS (pH 7.4), washing buffer (PBS containing 0.1% Triton X-100, for removing non-specific binding), 2×SDS loading buffer.

Core Reagents: Specific primary antibody (targeting the protein of interest), negative control antibody (e.g., irrelevant IgG, for excluding non-specific binding), Protein A/G magnetic beads/agarose beads (for capturing antibody-antigen complexes).

Equipment Preparation: Centrifuge, pipettes and tips, centrifuge tubes/EP tubes, magnetic rack (for magnetic beads), shaker, protein quantification kit (e.g., BCA kit).

Sample Pretreatment:

Cell Samples: Collect cells in the logarithmic growth phase, wash twice with pre-cooled PBS. After aspirating all PBS, add pre-cooled lysis buffer (100-200μL per 1×10⁶ cells) and lyse on ice for 30 minutes. Centrifuge at 12,000rpm at 4℃ for 15 minutes, then collect the supernatant (i.e., total protein extract). Determine the protein concentration using the BCA method.

Tissue Samples: Mince the tissue, add pre-cooled lysis buffer (500μL per 50mg tissue), homogenize, and lyse on ice for 30 minutes. Follow the same centrifugation step as for cell samples to collect the supernatant.

II. Antibody Conjugation with Protein A/G Beads (Formation of "Antibody-Bead" Complexes)

Take an appropriate amount of Protein A/G magnetic beads (20-50μL per 500μg total protein), add 500μL pre-cooled PBS, and gently invert to mix. After magnetically separating the beads (centrifuge agarose beads at 3,000rpm at 4℃ for 3 minutes to collect), discard the supernatant. Repeat washing twice.

Add the specific primary antibody to the washed beads (according to the manufacturer’s instructions, usually 1-5μg). For the negative control well, add an equal amount of irrelevant IgG. Supplement with PBS to a final volume of 200μL, and incubate slowly on a shaker at 4℃ for 1-2 hours (or at room temperature for 30 minutes) to ensure sufficient binding between the antibody and beads.

After incubation, wash the beads three times with 500μL washing buffer each time to remove unbound free antibody. Aspirate all supernatant thoroughly after the final wash and set aside for use.

III. Antigen Capture (Binding of Target Protein to "Antibody-Bead" Complexes)

Take the total protein extract (usually 200-500μg protein), adjust the volume to 500μL with lysis buffer, add the prepared "antibody-bead" complexes, and incubate slowly on a shaker at 4℃ for 4-6 hours (or overnight to ensure complete antigen binding).

After incubation, wash the complexes 4-5 times with 500μL washing buffer each time (magnetically separate magnetic beads or centrifuge agarose beads to collect). Aspirate the supernatant as thoroughly as possible after the final wash (to avoid residual impurities interfering with subsequent detection).

IV. Elution of Target Protein

Add 20-50μL 2×SDS loading buffer to the washed "antibody-bead-antigen" complexes and gently pipette to mix.

Heat in a boiling water bath for 5-10 minutes (or at 95℃ for 15 minutes) to dissociate the target protein from the complexes.

For magnetic bead samples: Magnetically separate the beads and collect the supernatant (containing the target protein). For agarose bead samples: Centrifuge at 12,000rpm at 4℃ for 5 minutes and collect the supernatant.

V. Subsequent Detection (Taking Western Blot as an Example)

Load the eluted protein samples onto an SDS-PAGE gel for electrophoretic separation.

After membrane transfer and blocking, incubate with a specific primary antibody against the target protein (which can be the same as the one used for IP or targeting a different epitope), followed by incubation with an HRP-conjugated secondary antibody.

Perform chemiluminescence and exposure to observe the target protein band (the negative control IgG group should show no obvious band to exclude non-specific binding).

Key Notes

All reagents must be pre-cooled, and all operations should be performed on ice or at 4℃ to minimize protein degradation.

Protease/phosphatase inhibitors must be added to the lysis buffer, and the type of inhibitors should be adjusted according to the target protein (e.g., phosphatase inhibitors are required for phosphorylated proteins).

Negative controls (irrelevant IgG) and positive controls (samples known to contain the target protein) should be set up simultaneously to ensure the reliability of experimental results.

Magnetic beads/agarose beads must be washed thoroughly to avoid residual free antibodies interfering with Western Blot results.