Journal
/
Biology
/
Affinity Nedbør av Active Rho-GEFs Bruke en GST-merket Mutant Rho Protein (GST-RhoA (G17A)) fra Epiteliale Cell lysates
JoVE Journal
Biology
This content is Free Access.
JoVE Journal Biology
Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Affinity Nedbør av Active Rho-GEFs Bruke en GST-merket Mutant Rho Protein (GST-RhoA (G17A)) fra Epiteliale Cell lysates

DOI:
10.3791/3932-v

•

11:28 min

•

March 31, 2012

•

, , , ,
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

Chapters

  • 00:05Title
  • 01:46Transformation of E. coli with the pGEX-RhoA(G17A) Construct
  • 02:55Preparation of GST-RhoA(G17A) Beads
  • 07:50GEF Pulldown Assay with Nucleotide-free RhoA(G17A) Beads
  • 10:11Representative Pull Down Results
  • 11:03Conclusion

Summary

Automatic Translation

Automatic Translation

Metoden som presenteres her beskriver en analyse for å følge aktivering av RhoA spesifikke BNP / GTP Utveksle Factors (GEFs) i dyrkede celler ved å gjøre bruk av en mutant RhoA GST fusion protein som har høy affinitet for aktivert GEFs. GEFs er fremskyndet fra celle lysates oppdaget ved Western blotting og kvantifisert ved densitometry.

Tags

Affinity PrecipitationRho-GEFsGST-tagged Mutant Rho ProteinCytoskeleton RegulationCellular ProcessesGTPasesGuanine-nucleotide Exchange FactorPathway-specific RegulationRegulatory NetworksActive Pool Of GEFsAffinity Precipitation AssayBurridge Lab

Article

Embed

ADD TO PLAYLIST

Usage Statistics

Related Videos

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

Kromatografiske Rensing av Highly Active gjær ribosomer

Ortogonale Protein Purification Tilrettelagt av en Small Bispecific Affinity Tag

Identifisering av Protein Samspill Partners Bruke Tandem Affinity Rensing

En væskefase Affinity Capture Assay hjelp av magnetiske perler å studere protein-Protein Interaction: Den poliovirus-Nanobody Eksempel

En protokoll for Fag Display og Affinitetsutvelgelse Bruke rekombinante protein agn

Transgen Gnagere analyse for Kvantifisering Mann Germ Cell Mutant Frequency

Read Article