Fig. 1. Generation and characterisation of anti-Geminin antibodies. (A) Coomassie staining of purified bacterially expressed His₆-Geminin. (B) Immunoblots of recombinant Geminin and human cell lysates with affinity-purified anti-Geminin antibodies G94 and G95. Antibodies G94 and G95 detected a single 33kDa protein in total cell lysates from asynchronous Jurkat, HeLa, MOLT-4 and HL-60 cells, and recognised nanogram quantities of recombinant Geminin (35 kDa) whereas preimmune (PI) sera did not. (C) Immunoprecipitation (IP) of HeLa S phase cell lysates with antibody G94 followed by immunoblotting (IB) with G95 shows the 33 kDa protein in the immunoprecipitate whereas immunoprecipitation with preimmune (PI) sera did not. (D) Bivariate flow cytometry of asynchronous MOLT-4 cells with antibody G95 alone, or G95 plus recombinant Geminin. Note that cells in G1 (2C) are Geminin-negative (depicted as area within the trapezoidal region) whereas cells in S or G2/M are positive (area outside the trapezoidal region). Preincubation of antibody G95 with recombinant Geminin blocked detection of this cell cycle specific expression.