Identification of SUV420H2 associated proteins using TAP/MS. (A) TAP-SUV420H2 purification from HeLa cells. TAP-SUV420H2 protein complexes were purified from HeLa cells, separated by SDS-PAGE, and stained with colloidal Coomassie. Some of the co-purified proteins identified by LC-MS/MS are indicated. (B) SUV420H2 peptide coverage is 36%. The identified peptides are indicated in red bold on the SUV420H2 protein sequence. Peptides are dispersed over the full sequence of the protein. (C) Western blotting showing the presence of HP1β in the TAP-SUV420H2 purification. Total protein extracted from HeLa cells expressing TAP-SUV420H2 (Whole lysate; 50 μg of proteins) and TAP purified sample (CBP Eluate; 20% of the material used for MS identification) were separated by SDS-PAGE and proteins detected by Western blotting using specific antibodies. Tagged SUV420H2 was revealed using an anti-CBP antibody (α-CBP), while HP1β was revealed by an anti-HP1β antibody (α-HP1β). Note that the TEV protease-mediated cleavage of the Protein A moiety of the TAP tag during the purification procedure increases the gel mobility of the tagged SUV420H2 protein in the CBP eluate as compared to whole extract.
Souza et al. BMC Cell Biology 2009 10:41 doi:10.1186/1471-2121-10-41