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Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates
JoVE Journal
Biologia
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JoVE Journal Biologia
Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

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11:28 min

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March 31, 2012

DOI:

10.3791/3932-v

DOI:
10.3791/3932-v

•

11:28 min
March 31, 2012

•

2 Views
, , , ,
1Keenan Research Centre, Li Ka Shing Knowledge Institute,St. Michael’s Hospital , 2Department of Surgery,University of Toronto, 3Department of Cell and Developmental Biology,University of North Carolina at Chapel Hill

Summary

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The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.

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Cite this Article

Waheed, F., Speight, P., Dan, Q., Garcia-Mata, R., Szaszi, K. Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates. J. Vis. Exp. (61), e3932, doi:10.3791/3932 (2012).

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