Rapid purification of nucleosome assembly protein (AP-I) and production of monoclonal antibodies against it

A nucleosome assembly protein (AP-I) was purified approximately 50% from the cytosol of HeLa S3 cells by three purification steps. Using this protein fraction as an antigen, we established three stable hybridomas that secrete monoclonal antibodies specific for AP-I by the conventional method of cell fusion. Immunoblotting of the HeLa S3 cytosol, proved AP-I exists as a 58-kDa peptide in vivo, not as the 53-kDa peptide previously identified as active in nucleosome assembly (Ishimi, Y., et al., Eur. J. Biochem., 142, 431-439, 1984). An immunocytochemical study using the monoclonal antibody with the highest specificity against AP-I pin pointed the intranuclear localization of AP-I in HeLa S3 cells.