Tandem Affinity Purification of Protein Complexes

TAP was performed as by Fernández et al. (2009) (link). Briefly, mouse forebrain was homogenized on ice in 1% deoxycholate (DOC) buffer (50 mM Tris [pH 9.0], 1% sodium deoxycholate, 50 mM NaF, 20 μM ZnCl₂, and 1 mM Na₃VO₄), 2 mM Pefabloc SC (Roche), and 1 tablet/10 mL protease inhibitor cocktail tablets (Roche) at 0.38 g wet weight per 7 mL cold buffer with a glass Teflon Douncer homogenizer. The homogenate was incubated for 1 hr at 4°C and clarified at 50,000 × g for 30 min at 4°C. TAP-tagged complexes were isolated as described previously (Fernández et al., 2009 (link)). The SDS-PAGE gel was fixed and stained with colloidal Coomassie, and lanes were cut into slices, destained, and digested overnight with trypsin (Roche, trypsin modified, sequencing grade) as described previously (Fernández et al., 2009 (link)). Peptide digestion, LC-MS/MS, and proteomics data analysis are described in the Supplemental Experimental Procedures.