Journal
/
Biology
/
Affinity udfældning af aktivt Rho-GEFs Brug en GST-mærket Mutant Rho Protein (GST-RhoA (G17A)) fra Epiteliale Cellelysaterne
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 udfældning af aktivt Rho-GEFs Brug en GST-mærket Mutant Rho Protein (GST-RhoA (G17A)) fra Epiteliale Cellelysaterne

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

Fremgangsmåden præsenteres her beskriver et assay til at følge aktivering af RhoA specifikke BNP / GTP Exchange faktorer (GEFs) i dyrkede celler ved anvendelse af en mutant RhoA GST-fusionsprotein, der har høj affinitet for aktiverede GEFs. GEFs udfældes fra cellelysater, detekteret ved Western blotting og kvantificeret ved densitometri.

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

Chromatografisk Rensning af højaktiv Gær ribosomer

Ortogonale Proteinoprensning Faciliteret af en lille Bispecific Affinity Tag

Identifikation af Protein Interagerende Partners Brug Tandem Affinitetsrensning

En væskefase affinitetsindfangning under anvendelse af magnetiske perler til at studere protein-protein interaktion: poliovirus-Nanobody Eksempel

En protokol for fagvisning og Affinity Selection Brug rekombinant protein Lokkemad

Transgene gnaver Assay til Quantifying Mand Kimcelle mutanthyppighed

Read Article