NIEHS Funds New Centers in Texas and California

Purified CenH3 IP material was electrophoretically separated on a 18-20% SDSPAGE gel, individual proteins were either visualized by Coomassie Brilliant Blue stain excised and subjected to an in-gel tryptic digest (digests were performed as described (Shevchenko et al, 1996), or the eluted CenH3 particles were precipitated with 10% trichloroacetic acid, washed once with acetone, dried, re-dissolved in de-ionized water, and digested in solution with Arg-C protease. For CenH3 identification, we could not detect peptides from the protein directly in the gel slice containing the tetramer (confirmed by Western blot analysis). Therefore, we used eluted CenH3 protein from extensively washed CenH3IP-beads.