substrate proteins were expressed with c - terminal his6 tags , and assayed in 50 mm tris , 50 mm k acetate , ph 8.0 , with 100 m sam as the methyl donor . all reactions were incubated 1 h at 30c and analyzed for methylation by immunoblot . for elisa assays , reactions were incubated as above then transferred to protein a - coated elisa plates pretreated with anti - me2-spk . after 1 h , the reaction was removed and wells were treated with 1% sds for 30 min . efficient removal of the initiating met is dependent on the identity of the second residue . to avoid this problem , we created fusions from residues 210 of rcc1 with his6-tag plus factor x cleavage site at the n - terminus , and c - terminal gfp . xpk - egfp substrate proteins were expressed in e.coli and purified on ni - nta beads , then cleaved using factor x. on - bead endoproteinase glu - c and asp - n digestions were used to identify the immunoprecipitated proteins . the retained n - termini were then eluted from the beads and analyzed for methylation ( supplementary fig . substrate proteins were expressed with c - terminal his6 tags , and assayed in 50 mm tris , 50 mm k acetate , ph 8.0 , with 100 m sam as the methyl donor . all reactions were incubated 1 h at 30c and analyzed for methylation by immunoblot . for elisa assays , reactions were incubated as above then transferred to protein a - coated elisa plates pretreated with anti - me2-spk . after 1 h , the reaction was removed and wells were treated with 1% sds for 30 min . efficient removal of the initiating met is dependent on the identity of the second residue . to avoid this problem , we created fusions from residues 210 of rcc1 with his6-tag plus factor x cleavage site at the n - terminus , and c - terminal gfp . xpk - egfp substrate proteins were expressed in e.coli and purified on ni - nta beads , then cleaved using factor x. on - bead endoproteinase glu - c and asp - n digestions were used to identify the immunoprecipitated proteins . the retained n - termini were then eluted from the beads and analyzed for methylation ( supplementary fig .