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Rab GTPases pp 47-60 | Cite as

High-Throughput Assay for Profiling the Substrate Specificity of Rab GTPase-Activating Proteins

  • Ashwini K. Mishra
  • David G. LambrightEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1298)

Abstract

Measurement of intrinsic as well as GTPase-Activating Protein (GAP)-catalyzed GTP hydrolysis is central to understanding the molecular mechanism and function of GTPases in diverse cellular processes. For the Rab GTPase family, which comprises at least 60 distinct proteins in humans, putative GAPs have been identified from both eukaryotic organisms and pathogenic bacteria. A major obstacle has involved identification of target substrates and determination of the specificity for the Rab family. Here, we describe a sensitive, high-throughput method to quantitatively profile GAP activity for Rab GTPases in microplate format based on detection of inorganic phosphate released after GTP hydrolysis. The method takes advantage of a well-characterized fluorescent phosphate sensor, requires relatively low protein concentrations, and can in principle be applied to any GAP-GTPase system.

Key words

GTPase Rab GTPase Phosphate-binding protein Phosphate PBP-MDCC GTP hydrolysis GAP reaction High throughput GAP assay 

Notes

Acknowledgements

This work was supported by an NIH grant GM056324 to DGL.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular Pharmacology, Program in Molecular MedicineUniversity of Massachusetts Medical SchoolWorcesterUSA

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