Mass spectrometry identifies proteins that interact with HDAC2 in the KOA model

CO-IP experiment pulls proteins that interact with HDAC2 protein in KOA model and performs mass spectrometry1. Sample whole protein extraction: induce KOA cell model. Digest and collect cells. Centrifuge and discard supernatant to collect precipitate. Add pre-cooled lysis buffer, vortex and mix to completely lyse at room temperature. Centrifuge conditions: 20000g, 4℃. Centrifuge for 20min to collect protein supernatant and store in -80℃ refrigerator.2. Use BCA kit to determine protein concentration, and divide samples into input, HDAC2-IP group, and NC group.3. Protein immune complex preparation: Take 5ul HDAC2 antibody stock solution and add it to HDAC2-IP histone supernatant, take 5ul IgG liquid and add it to NC histone supernatant, slowly invert and mix at 37℃ for 4h, so that the target protein and the interacting side are immunoprecipitated from the IP histone supernatant by HDAC2 specific antibody, and the NC group is used as a control.4. Preparation of protein A/G agarose beads: Take 100 µl protein A/G agarose beads, add 200ul CO-IP buffer and wash 3 times with a pipette. After each wash, centrifuge at 4℃, 3000 g for 30 s to retain the agarose beads. Add 100ul CO-IP buffer to resuspend, block with 4% BSA blocking solution for 1h, then centrifuge at 4℃, 3000 g for 30 s to retain the agarose beads. Resuspend and wash protein A/G agarose beads 3 times with PBS. During the last wash, transfer the liquid and protein A/G agarose beads to a new EP tube, then centrifuge at 4℃, 3000 g for 30 s to retain the agarose beads.5. Capture immune complex: Add the protein immune complex prepared in step 3 to the EP tube containing protein A/G agarose beads, fill the system with CO-IP buffer to 1ml, mix and incubate at 4℃ for 2h; then centrifuge at 4℃, 1000g for 1min, take the precipitate and add 200ul wash buffer, wash 2-3 times; centrifuge again to retain the precipitate.6. Elution to prepare protein spectrum sample: Add 40ul RIPA and 10ul 5X loading buffer, mix well, boil in a metal bath at 100℃ for 5min, store the sample in dry ice, and send for mass spectrometry.