An In Vitro System to Evaluate the Scaffold Function of the RalA Effector Protein RalBP1

The in vitro kinase assay is an invaluable method to identify and characterize kinase substrates and to determine specific site(s) of phosphorylation. However, the use of recombinant proteins does not adequately account for the contribution of accessory proteins, such as scaffolds, that can contribute to the phosphorylation reaction in vivo. We describe here a method to evaluate the contribution of specific scaffold proteins to kinase reactions using a modified in vitro kinase assay.