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.
Ishimi
Y
Y
Sato
W
W
Kojima
M
M
Sugasawa
K
K
Hanaoka
F
F
Yamada
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D018797:Cell Cycle Proteins; D002459:Cell Fusion / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000032:analysis*; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D008970:Molecular Weight; D009687:Nuclear Proteins; D056490:Nucleosome Assembly Protein 1; D009707:Nucleosomes / Q000032:analysis*; D011506:Proteins / Q000276:immunology / Q000302:isolation & purification*; D013194:Staining and Labeling
D000911:Antibodies, Monoclonal; D018797:Cell Cycle Proteins; C533557:NAP1L1 protein, human; C533558:Nap1l1 protein, mouse; D009687:Nuclear Proteins; D056490:Nucleosome Assembly Protein 1; D009707:Nucleosomes; D011506:Proteins