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. 2015 Mar 17;290(19):12355–12369. doi: 10.1074/jbc.M114.625012

FIGURE 1.

FIGURE 1.

Characterization of a monoclonal antibody targeting human Orc1. A, Coomassie Brilliant Blue-stained gel showing step for purification of the Orc1-ΔN400 fragment from E. coli cells. B, epitope mapping for the Orc1 antigen. The fragments of MBP-fused Orc1 were subjected to immunoprecipitation (IP) using the Orc1 78-1-172 monoclonal antibody, and then Western blots were performed with anti-MBP antibody. Only the full-length (FL) MBP-Orc1 and MBP-Orc1-ΔN400 versions but not other N-terminal truncated mutants were immunoprecipitated. C, silver-stained gel showing that increasing amounts of beads cross-linked to Orc1 78-1-172 monoclonal antibody, but not IgG, can immunoprecipitate endogenous Orc1 protein as well as the exogenously expressed GFP-Orc1 fusion protein from HeLa cells expressing GFP-Orc1. D, immunoblot detecting Orc1 and GFP in immunoprecipitates similar to those shown in C. E, Orc1 78-1-172 monoclonal antibody can co-immunoprecipitate other subunits of ORC, including Orc3 and Orc4 as detected by immunoblot.