Anti MCM2

Western blot using Rockland's Affinity Purified anti-MCM2 antibody shows detection of both phosphorylated and unphosphorylated MCM2 present in nuclear extracts from elutriated human cells (MO59K/K562). The MCM2 protein is phosphorylated after initiation of DNA replication, therefore, the protein is unphosphorylated in early S phase, and gradually becomes phosphorylated throughout S phase. In G2/M, all MCM2 is phosphorylated. Panel A shows western blot results for lysates were prepared from asynchronous cells (lane 1), cells arrested in early S with aphidicolin (lane2), and cells arrested in mitosis with nocodazole (lane 3). Panel B shows a schematic diagram of bands representing phosphorylated and unphosphorylated MCM2 present in these preparations. Asynchronous cells contain a doublet of both forms. Aphidicolin treated cells contain only unphosphorylated MCM2 and nocodazole treatment results in only phosphorylated MCM2 detected in the lysate. The phosphorylated band migrates faster than the unphosphorylated form and is seen as the lower band. There is a clear switch from the unphosphorylated form in the center lane, to the phosphorylated form in the third lane, confirming recognition of both forms of MCM2 by this antibody. The primary antibody was diluted 1:400 for this experiment. Personal Communication, Jennifer Seiler, NIH, CCR, Bethesda, MD.