Co-Immunoprecipitation

Co-immunoprecipitation (Co-IP): Co-immunoprecipitation technology, based on the specific immune reaction between antibodies and antigens, is a classic method for verifying protein interactions. The special feature of Co-IP is that it can determine the natural binding of two proteins in intact cells, the detection results are highly reliable, and it is a powerful tool for constructing protein dynamic networks.

Technical principle: Lyse intact cells under non-denaturing conditions to obtain the protein interaction state existing in the cells. If pre-immobilized antibody protein to protein A on agarose-beads. After adding the beads, not only protein A can be immunoprecipitated, but protein B bound to protein A in the cell will also be precipitated together. After elution, the precipitated complex can be further detected by Western blot to confirm the interaction between the two proteins, or to quantitatively/qualitatively analyze the content of specific antigens in the sample^[1].

Development and application: With the development of co-immunoprecipitation technology, immunoprecipitation technology can be cross-linked with mass spectrometry (ie. IP-MS) to distinguish real protein-protein interactions from background noise. In fact, in immunoprecipitation experiments, background noise is often caused by the production of non-specific binders. These include agarose, agarose or (hydrophilic) polymer coated magnetic beads, or proteins bound to beads or fusion tags. Therefore, the target protein captured by the "bait" of the beads needs to undergo strict elution conditions to ensure the authenticity of the precipitate. However, due to strict operation, proteins with weak binding force to antibodies will be missed. If the precipitate is digested to become a peptide for mass spectrometry analysis, the real protein-protein interaction complex can be distinguished by comparing the ratio between the heavy peak and the light peak of the protein^[2].

Co-IP experiments are complex and require not only high-quality antibodies but also experienced operating skills. MCE provides you with high-quality immunoprecipitation-specific reagents and professional technical support services. For more service information, please visit the official website: https://www.medchemexpress.com/

MCE has not independently verified the accuracy of these methods. They are for reference only.

Co-IP experiments identify protein complexes formed by direct or indirect interactions. Here, we’ll describe the Co-IP process and common problems^[3][4][5].

1. Gel Preparation

(1) Reagents: 10% ammonium persulfate solution, 1.5 M Tris-HCl (pH=8.8), 1.0 M Tris-HCl (pH=6.8), 10% SDS, 5× electrophoresis buffer stock solution, transfer buffer , TBST buffer, blocking solution (store at 4°C).

(2) Prepare protein gel: Assemble the instrument as required and fix it, seal the bottom of the glass plate with 1% Agarose. Prepare respectively protein separation gel (3/4 height of flat glass plate) and protein concentration gel (height of flat glass plate). See the attachment for the preparation system.

2. Operation process

(1) Pre-preparation of reagents: Pre-cooled PBS buffer, RIPA buffer, cell scraper (wrapped with plastic wrap and buried under ice), centrifuge. Add the protease inhibitor PMSF* (HY-B0496) in advance in the RIPA buffer.

*: Aqueous solutions of PMSF are very unstable. Half of it will be degraded in 30 minutes. It is recommended to use it immediately and use it as soon as possible.

(2) Total protein extraction of cell samples: Harvest cells 24-48 h after transfection, rinse with PBS, and repeat twice. Discard PBS last time. Add to the culture bottle 5x volume of pre-cooled RIPA lysate (about 250-350 μL). Spread the lysate repeatedly and react on ice for 30 min. Scrape the cells with a cell scraper, pipette the cell suspension into a 1.5 mL centrifuge tube, and continue blowing to break the cells. Centrifuge at 12,000 g for 30 min at 4°C , and collect the supernatant of the cell lysate. Transfer the supernatant to a new centrifuge tube with a pipette.

(3) Determination of protein concentration: Use the Bradford method to develop a protein standard curve and determine the protein concentration. The total protein was diluted at least before the assay 10 to reduce the impact of detergent in the cell lysate (after aliquoting, it can be -20°C stored for one month). Use PBS to dilute the total protein to approx. 1 μg/μL to reduce the concentration of detergent used in the lysate. If the protein of interest is low in the cells, the total protein concentration should be slightly above this value (e.g. 10 μg/μL).

(4) Preparation of solid-phase adsorption matrix: Here will use Protein A Agarose Beads (HY-K0213) and Protein A Magnetic Beads (HY-K0203) for example*. Before using these two solid-phase matrices, apply rinse with PBS and repeat twice (3000 rpm, 3 min each time). The purpose is removing non-specific impurities and reducing background noise. After rinsing, Protein A Agarose Beads needs to be added with the PBS to prepare 50% concentrated suspension. It is recommended to reduce the tip of the pipette tip to facilitate the absorption of Protein A Agarose Beads.

*: Agarose beads are easy to use, large diameter, strong binding force, porous and easy to adsorb; Magnetic beads have small diameter, low background, less antibody consumption, and need to use a magnetic frame during operation.

(5) Immunoprecipitation: If using Beads immobilized with antibodies and Protein A by cross-linking agent, mix 100 μL (50% concentration) of agarose beads or 10 μL of magnetic beads per 1 mL of total protein. Ratio, add protein extract to Beads. 4°C, shake horizontally for 10 min. If you are using unfixed antibody Beads, you should add a certain volume of rabbit antibody to the extract. The dilution ratio of the antibody in the total protein is based on experience. 4°C, shake the antigen-antibody mixture slowly overnight or at room temperature 2 h (recommended for kinase or phosphatase activity assays 2 hours incubate at room temperature). Lastly, add 100 μL Protein A Agarose Beads to pull down the antigen-antibody complex. 4°C, shake the antigen-antibody complex slowly overnight (or under room temperature for 1 h). If use mouse or chicken antibody, it is recommended to add 2 μL "transitional antibody" (rabbit anti-mouse IgG or rabbit anti-chicken IgG).

(6) Collection and processing of precipitated complexes: a. Collect Agarose beads-antigen-antibody complexes. Centrifuge at 3000 rpm for 3 min at 4°C, centrifuge the Agarose beads to the bottom of the tube, and carefully aspirate the supernatant. b. Collect the Magnetic bead-antigen-antibody complex. 4°C, use a magnetic stand to absorb the magnetic beads to the bottom of the tube, and use a pipette to remove the extract. Re-add 800 µL pre-cooled RIPA buffer* to wash the complex, repeat 3 times. Finally, add 60 μL of 2x SDS-PAGE Buffer, mix gently, and resuspend the Beads-antigen-antibody complex. The amount of buffer can be determined according to the needs of loading. The amount of 60 μL is sufficient for three loadings.

*: The range of RIPA buffer used in this step is limited, which may destroy the internal binding of the agarose bead-antigen-antibody complex. Accounting for certain situation, RIPA buffer can be replaced with PBS buffer for washing.

(7) Pretreatment for immunoassay: Place the loaded sample in a water bath at 99°C for 5 minutes (add explosion-proof clips to each sample tube) to free antigen, antibody, and beads. Immediately after the water bath, place on ice for later use. Centrifuge, collect the supernatant for electrophoresis (can be temporarily frozen at -20°C), and collect the remaining Protein A agarose beads. The supernatant should be reboiled before electrophoresis 5 minutes transsexual.

3. Determination of Bound Proteins by Co-immunoprecipitation

(1) Add the sample to the discontinuous SDS-PAGE in gradient gel, electrophoresis overnight at a constant current with 10 mA;

(2) Observe protein bands by Brilliant Blue staining;

(3) Cut the target band from the gel, put it into a microcentrifuge tube, and wash twice with 1 mL 50 % acetonitrile, each time 3 minutes;

(4) Digest the protein in the gel with trypsin, and then separate the peptide by electroelution;

(5) Separate peptides by narrow-bore high-performance liquid chromatography. Edman degradation sequencing is performed on ABI 477A or ABI 494A Protein sequencer.

1. Cell lysis: Use mild conditions to lyse the cells to ensure that all protein-protein interactions in the cells are completely preserved; generally use a non-ionic denaturant, such as Triton X-100 (HY-Y1883A); the lysis of each cell The conditions are different and should be determined empirically; avoid the use of high concentrations of denaturants (0.2 % SDS); do not omit enzyme inhibitors in the cell lysate.

2. Antibody selection: Use antibodies with a clear source; multiple antibodies can be used together. Commonly used control antibodies, such as monoclonal antibodies: of normal mice IgG or another monoclonal antibody; rabbit polyclonal antibody: normal rabbit IgG.

3. Avoid background contamination: It is necessary to distinguish specific precipitated proteins from exogenous non-specific proteins, and the use of monoclonal antibodies can help avoid contamination. A control group can be set up to obtain precipitated complexes in cell lysate that do not express antigens and compare them with the experimental group to select products with specific protein peaks.

4. Experimental background: Carry out subcellular localization and determine protein-protein interaction sites. The CO-IP experiment is suitable for the verification of the interaction in cells, but not for the interaction caused by cell lysis.

5. Gel preparation details: Prepare protein separation gel, and the order of adding samples is from top to bottom. After adding the sample, mix quickly and use 5-mL pipette to add the liquid between the two glass plates. It should be perfused at a constant speed to avoid air bubbles, and stop when the perfusion reaches 3/4 of the flat glass plate. After perfusion of the separation gel, add ddH₂O to the edge of the glass plate within 30-60 s and let it stand for 30 min until the separation gel and the water layer have a clear boundary. Absorb residual liquid with absorbent paper. Configure protein stacking gel, add samples from top to bottom, mix quickly after adding samples and use 5-mL pipette to add the liquid between the two glass plates at a constant speed to avoid air bubbles and stop when it reaches the top of the flat glass plate. Immediately insert the mold comb after adding the sample, and use it after it is completely solidified (approx. 30 minutes).