Anti his antibody 631212

To examine APC/C activity towards securin the ubiquitination assay was performed with 60 nM of recombinant human APC/C, 150 nM UBA1, 300 nM UbcH10, 300 nM Ube2S, 20 μM ubiquitin, 2 μM securin, 5 mM ATP, 0.25 mg/mL BSA and 7 nM of recombinant human Cdc20. The ubiquitination products of securin were detected by western blot with either an anti-His antibody (631212; Clontech) or an anti-securin antibody (700791; Invitrogen).
To test the activity of a pre-assembled APC/C^MCC complex towards Cdc20^MCC (Fig. 5c), ubiquitination reactions were performed with 250 nM of recombinant human APC/C^Cdc20-MCC and 10 μM of UbcH10 (40x excess). To test the activity of APC/C towards the Cdc20^MCC from individually purified wild type and mutant MCC^{BubR1-StrepII} (purification by StrepII^x2 affinity and gel filtration columns) ubiquitination reactions were performed with 200 nM of recombinant human APC/C^OA, 200 nM of recombinant human Cdc20 and either 300 or 600 nM of recombinant human MCC^{BubR1-StrepII} (Fig. 5d, e). Either with a pre-assembled APC/C^MCC complex or with a molar excess of MCC complex over free Cdc20 and APC/C only Cdc20^MCC ubiquitination is promoted (data not shown)20 (link). Cdc20 and the ubiquitination products of Cdc20^MCC were detected by western blot with an anti-Cdc20 antibody (Cdc20 H-175 sc-8358; Santa Cruz Biotechnology, Inc.).