Principle:Co-Im muno precipitati on (Co-IP) was developed from the immun oprecipitati on tech nique with which Co-IP shares the fun dame ntal pr in ciple of the specific an tige n-an tiody reacti on. Co-IP helps determ ine whether two prote ins in teract or not in physiological conditions in vitro. Graphically, the Co-IP principle is as described in the right hand side picture.The known prote in (an tige n) is termed the bait prote in, and the prote in it in teracts with is called the prey prote in. The sta ndard Co-IP protocol is the same as that described for IP, and actually any system desig ned for IP should also work for Co-IP.After that cells are completely lysed un der non-den aturi ng con diti ons, prote ins that bound together are kept. Therefore if you use an ti-X to precipitate prote in X through Co-IP, the n you can get other prote ins that in teract with protein X in situ.Co-IP is applied to test whether two known prote ins bind each other in cells, or to find a new protein that in teracts with a known protei n.Advantages:1. Protei ns that in teract in a typical Co-IP are post-tra nslatio nally modified and con formatio nally n atural.2. In Co-IP protei ns in teract in a non-den aturi ng con diti on which is almost physiological.Disadvantages:1. The signals of low-affinity of protein interactions might not be detected.2. There might be a third protein in certain protein-protein interaction.3. To choose an appropriate antibody, the target protein needs to be properly predicted. Or there would not be a positive result in Co-IP.Reagents and buffers: PBS RIPA (RadioIm mun oPrecipitati on Assay) Lysis buffer:Tris-HCl: 50 mM, pH 7.4No nidet P-40 (NP-40): 1%Deoxycholate Na: 0.25%NaCl: 120 mMEDTA: 1 mM*PMSF: 1 mM* Leupep tin 1 “g/ml*Aproti nin 1 pg/ml*Pepstati n1 pg/ml*Na3VO4： 1 mM*NaF: 1 mMNote: In gredie nts labeled with * should be added right before each use. PMSF degrades to a half after 30min in water. Wash ing buffer :lysis buffer:5M NaCl =100:1 (ensure NaCl not exceed 1M) prote in A/G-agarose beads Specific an tibody (MAb or PAb)Protocol:DAY 11. 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 4C.4. Centrifuge at 14,000 g 4OC 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 worki ng soluti on (in PBS)6. Add in 50% protein A/G agarose with ratio of 100pl for a 1ml sample solutio n. Shake on horiz on tal shaker for 10min, 4C (This step aims to elimi nate non-specific bi ndi ng protei ns)7. Centrifuge 14,000g at 4OC for 15min, transfer the supernatant to new tubes and discard prote in A/G-agraose beads8. Quantify total protein with BCA assay or other methods.9. Dilute the total protein to 1pg/pl with PBS to decline the concentrations of detergents. If you feel the concentration of your target protein is low, you can dilute the total prote in to 10pg/pl. (if its high eno ugh)10. Add in appropriate amount of primary antibody to approximately 500pl total volume.11. Slowly shake antigen-antibody complex on rotating shaker at 4C for over ni ght.Note: if dow nstream experime nt is en zyme activity assay for kin ase or phosphatase, its better to change step 11 to a 2h incubation at room temperature.DAY 212. Centrifuge 14,000g for 5s, keep the pellet and wash with pre-chilled washing buffer (or cold PBS) for 3 times. (800pl each)13. Collect the supernatant to proceed to SDS-PAGE, western-blot, or mass spectra an alysis.Note： This Co-IP protocol is to bind an tibody to the Prote in A/G-argarose beads and the n mix with the an tige n. It gives lesser yield tha n the other one and avoids the problem of co-eluti on of an tibodies. If you want to yield high purity of target prote in regardless of non-specific binding, you can mix an tibody with prote in sample prior to additi on of Prote in A/G-agarose beads, thus in the end the an tibodies are also co-eluted with target prote in and interference might occurs in western blot detection.