Rb-C (retinoblastoma protein C-terminus) Fusion Protein serves as a useful substrate for various cyclin-dependent kinases (CDK's) (8,9,11). It is expressed as a recombinant protein fusion of Rb residues 701-928 and maltose binding protein. The phosphorylation sites present in this C-terminal portion of Rb include Ser780 (8), Ser795 (9) and Ser807/811 (11).
Apparent Molecular Weight: 68 kDa
Rb-C Fusion Protein at a concentration of 2 µg/20 µl reaction can be phosphorylated using cdc2 Protein Kinase (NEB #P6020) in an in vitro kinase assay with 1X Kinase Buffer #9802 and 200 µM ATP #9804. After 30 minutes at 30ºC, phosphorylation can be detected by Western blot with Phospho-Rb (Ser795) Antibody #9301.
The purified protein was resolved on two identical SDS-polyacrylamide gels. One was stained with Coomassie brilliant blue and the other was blotted to PVDF membrane and the protein band detected using Rb antibody. Greater than 95% of the observable protein was identified as the Rb-C Fusion Protein by apparent molecular weight (68 kDa), and only one band was identified by immunoblotting.
Recombinant protein fusion of Rb residues 701-928 and maltose binding protein. Purified by the pMAL Protein Purification System (New England Biolabs #E8000S).
The retinoblastoma tumor suppressor protein Rb regulates cell proliferation by controlling progression through the restriction point within the G1-phase of the cell cycle (1). Rb has three functionally distinct binding domains and interacts with critical regulatory proteins including the E2F family of transcription factors, c-Abl tyrosine kinase, and proteins with a conserved LXCXE motif (2-4). Cell cycle-dependent phosphorylation by a CDK inhibits Rb target binding and allows cell cycle progression (5). Rb inactivation and subsequent cell cycle progression likely requires an initial phosphorylation by cyclin D-CDK4/6 followed by cyclin E-CDK2 phosphorylation (6). Specificity of different CDK/cyclin complexes has been observed in vitro (6-8) and cyclin D1 is required for Ser780 phosphorylation in vivo (9).
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