Co-IP

Co-Immunoprecipitation, Co-IP

Principle

Reagents and buffers

Protocol

Applications of Co-IP

Co-IP FAQ

Protocol

Principle:

Co-Immunoprecipitation (Co-IP) was developed from the immunoprecipitation technique with which Co-IP shares the fundamental principle of the specific antigen-antiody reaction. Co-IP helps determine whether two proteins interact or not in physiological conditions in vitro. Graphically, the Co-IP principle is as described in the right hand side picture.

The known protein (antigen) is termed the bait protein, and the protein it interacts with is called the prey protein. The standard Co-IP protocol is the same as that described for IP, and actually any system designed for IP should also work for Co-IP.

After that cells are completely lysed under non-denaturing conditions, proteins that bound together are kept. Therefore if you use anti-X to precipitate protein X through Co-IP, then you can get other proteins that interact with protein X in situ.

Co-IP is applied to test whether two known proteins bind each other in cells, or to find a new protein that interacts with a known protein.

Reagents and buffers:

∙ PBS

∙ RIPA (RadioImmunoPrecipitation Assay) Lysis buffer:

Note: Ingredients labeled with * should be added right before each use. PMSF degrades to a half after 30min in water.

∙ Washing buffer :

lysis buffer:5M NaCl =100:1 (ensure NaCl not exceed 1M)

∙ protein A/G-agarose beads

∙ Specific antibody (MAb or PAb)

Protocol:

DAY 1

1. Carefully wash cultured cells with pre-chilled PBS for 2 times.

2. Add in cold RIPA lysis buffer (1ml for 107cells).

3. Scrap cells off to clean 1.5ml eppendorf tubes with a clean, cold scraper. Put them on a low-speed rotating shaker for 15 min at 4°C.

4. Centrifuge at 14,000 g 4°C for 15min, transfer the supernatant to new tubes immediately.

5. Wash protein A/G-agarose beads for 2 times with PBS and make a 50% protein A/G agarose working solution (in PBS)

6. Add in 50% protein A/G agarose with ratio of 100μl for a 1ml sample solution. Shake on horizontal shaker for 10min, 4°C (This step aims to eliminate non-specific binding proteins)

7. Centrifuge 14,000g at 4°C for 15min, transfer the supernatant to new tubes and discard protein A/G-agraose beads

8. Quantify total protein with BCA assay or other methods.

9. Dilute the total protein to 1μg/μl with PBS to decline the concentrations of detergents. If you feel the concentration of your target protein is low, you can dilute the total protein to 10μg/μl. (if it’s high enough)

10. Add in appropriate amount of primary antibody to approximately 500μl total volume..

11. Slowly shake antigen-antibody complex on rotating shaker at 4°C for overnight.

Note: if downstream experiment is enzyme activity assay for kinase or phosphatase, it’s better to change step 11 to a 2h incubation at room temperature.

DAY 2

12. Centrifuge 14,000g for 5s, keep the pellet and wash with pre-chilled washing buffer (or cold PBS) for 3 times. (800μl each)

13. Collect the supernatant to proceed to SDS-PAGE, western-blot, or mass spectra analysis.

Note: This Co-IP protocol is to bind antibody to the Protein A/G-argarose beads and then mix with the antigen. It gives lesser yield than the other one and avoids the problem of co-elution of antibodies. If you want to yield high purity of target protein regardless of non-specific binding, you can mix antibody with protein sample prior to addition of Protein A/G-agarose beads, thus in the end the antibodies are also co-eluted with target protein and interference might occurs in western blot detection.

References:

∙ Ohh M. et al. (1998) The von hippel-lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix. Mol Cell. 1, 959-68.

∙ Golemis E. (2002) Protein-protein interactions : A molecular cloning manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. ix, 682.

Applications of Co-IP

Applications of Co-Immunoprecipitation, Co-IP: to test the proteins’ interactions

∙ Find a new protein that interacts with a known one:

After typical Co-IP, identify the new protein that binds with a known protein by western-blot or (and) mass spectrometry.

∙ Prove a interaction between target protein and tested protein:

After typical Co-IP, perform western blot test with tested protein’s antibody to prove its existence. Thus tested protein is proved to interact with known protein.

Co-IP FAQ

◆What’s the difference between Co-IP and GST-pull down?

Co-immunoprecipitation experiment is processed under non-denaturing conditions which can preserve natural activity of interactions between proteins to the largest content. Therefore, Co-IP can well reflect the real protein interaction in situ. However, only with Co-IP we can’t decide whether the protein interaction is direct or indirect. After co-immunoprecipitation by antibodies of target protein, we get a protein complex which not only contains proteins that directly interact with target protein.

For GST-pull down test, we can change experimental conditions in vitro to eliminate effects of non-relevant proteins. Thus we can easily make sure whether target protein interacts with tested protein or not.

◆I experience non-specific binding in my immunoprecipitation experiment.

∙ Use more stringent washing buffer for washing.

∙ Add a non-ionic detergent (Tween-20 or Triton X-100) to the washing buffer, in concentrations between 0.01-0.1.

∙ If the beads are blocked before precipitation, add identical blocker to the washing buffer.

∙ Increase the number of washing steps or prolong the washing steps.

∙ Decrease incubation time (beads and sample).

∙ Decrease the antibody concentration.

∙ A pre-clearing step is prefered to remove molecules that non-specifically bind to the protein A/protein G or the beads themselves.

Co-immunoprecipitation,Co-IP Troubleshooting

◆What if I got no protein that interacts with my target protein? Or, what if the signal of interactive protein was too weak?

That may be due to these reasons:

∙ The detergent in the lysis buffer might had too high a concentration or too strong.

∙ Decrease the concentration of the detergent, or change it to a mild one. Typically, a strength order of detergents should be:

∙ SDS>Trition>NP40>Digitonin>CHAPS

∙ Effect of sub-localization of your target protein in cells.

∙ Change the recipe of the lysis buffer and make the protein released.

∙ The protein-protein interactions were too weak or unstable.

Change the antibody to a more affinitive one to capture more of target protein.

Or, apply a expression system to enhance the purity and concentration of target protein.

Or, change origins of samples or optimize the sample treatment to let the sample have more of target protein or interactive proteins.

◆What if the false positives were too strong or too many non-specific binding proteins were detected?

That may be due to these reasons:

∙ The antibody concentration was too high.

∙ Low down the antibody concentration

∙ The antibody specificity was not good enough.

∙ Change the antibody to a better one.

If the non-specific binding persists, increase the concentration of NaCl to lower than 1M.

◆I seem to get low binding of my selecting antibody. What can I do to improve binding?

∙ Verify binding/specificity of your antibody to your antigen, e.g., by ELISA.

∙ Check the binding of your antibodies to the beads. If the antibodies are not captured and bound to the beads, the immunoprecipitation experiment will not work.

∙ Check the amount of beads and sample volume. With reference to the capacity of different beads proposed in the package inserts, increase the amount of beads or the concentration of your antibody during coupling.

∙ Increase the incubation time.

∙ Try another antibody.

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