WASP protein VCA domain: GST tagged: human

WASP protein VCA domain: GST tagged: human
$0.00

Product Uses Include

  • Stimulating Arp2/3 complex activity
  • Isolating Arp2/3 complex from cell or tissue extract

Material
The VCA (Verprolin, cofilin, acidic) domain of human WASP protein has been expressed in a bacterial expression system as a GST-tagged fusion protein. The protein has been purified by glutathione affinity chromatography and is supplied as a lyophilized powder. When reconstituted with distilled water, the complex is in the following buffer: 20 mM Tris pH 7.5, 25 mM KCl, 1 mM MgCl2, 0.5 mM EDTA, 0.2% dextran, 2% sucrose. The molecular weight of the GST-VCA domain protein is approximately  43 kDa.
 

Purity
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are >95% pure (Figure 1).

vcg03gel

Figure 1: Purity determination of GST-WASP VCA domain. 20 µg of VCG03 was run on an SDS-PAGE gel and visualized by coomassie blue staining. Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat.# ADV02).

Biological Activity
VCG03 was tested in an actin polymerization assay (Cat. # BK003). In conjunction with Arp2/3 (Cat. # RP01) it was shown to stimulate actin polymerization 20-fold compared to the control without VCG03. This indicates VCA domain specificity for stimulating the actin nucleating activity of Arp2/3.

arp-actin-pol

Figure 2: Actin polymerization stimulated by Arp2/3 complex and the VCA domain of WASP. Actin polymerization was measured using kit BK003. The addition of Arp2/3 complex or the VCA domain alone to actin has minimal effects on actin polymerization, while the combination of Arp2/3 and the VCA domain strongly stimulates the rate of actin polymerization. 

For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com

AuthorTitleJournalYearArticle Link
Nakamura, Hideki et al.ActuAtor, a Listeria-inspired molecular tool for physical manipulation of intracellular organizations through de novo actin polymerizationCell reports2023ISSN 2211--1247
Cao, Muqing et al.An actin filament branching surveillance system regulates cell cycle progression, cytokinesis and primary ciliogenesisNature Communications 2023 14:12023ISSN 2041--1723
Bogucka-Janczi, Katarzyna et al.ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization.eLife2023ISSN 2050--084X
Bock, Fabian et al.Rac1 promotes kidney collecting duct integrity by limiting actomyosin activityJournal of Cell Biology2021ISSN 1540-8140
Chen, Yuewen et al.Coronin 2B regulates dendrite outgrowth by modulating actin dynamicsFEBS Letters2020ISSN 1873-3468
Lin, Shan Shan et al.Dynamin-2 Regulates Postsynaptic Cytoskeleton Organization and Neuromuscular Junction DevelopmentCell Reports2020ISSN 2211-1247
Markwell, Steven M. et al.Cortactin phosphorylation by casein kinase 2 regulates actin-related protein 2/3 complex activity, invadopodia function, and tumor cell invasionMolecular Cancer Research2019ISSN 1557-3125
Karimi, Shaghayegh Sadr et al.Carbon nanotubes as molecular transporters to study a new mechanism for molecular entry into the cell nucleus using actin polymerization forcePLoS ONE2019ISSN 1932-6203
Schaffer, Ashleigh E. et al.Biallelic loss of human CTNNA2, encoding αN-catenin, leads to ARP2/3 complex overactivity and disordered cortical neuronal migrationNature Genetics2018ISSN 1546-1718
Tu, Ye et al.EFhd2/swiprosin-1 regulates LPS-induced macrophage recruitment via enhancing actin polymerization and cell migrationInternational Immunopharmacology2018ISSN 1878-1705
Mikhaylova, Marina et al.Caldendrin Directly Couples Postsynaptic Calcium Signals to Actin Remodeling in Dendritic SpinesNeuron2018ISSN 1097-4199
Zhao, Miao et al.Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerizationOncotarget2017ISSN 1949-2553
Rondina, M. T. et al.Non-genomic activities of retinoic acid receptor alpha control actin cytoskeletal events in human plateletsJournal of Thrombosis and Haemostasis2016ISSN 1538-7836
San Ruiz-Miguel, José E. et al.The role of Arp2/3 in growth cone actin dynamics and guidance is substrate dependentJournal of Neuroscience2014ISSN 1529-2401
Chan, Eddie et al.The acetylenic tricyclic bis(cyano enone), TBE-31 inhibits non-small cell lung cancer cell migration through direct binding with actinCancer Prevention Research2014ISSN 1940-6215
Xavier, Charles Peter et al.Phosphorylation of CRN2 by CK2 regulates F-actin and Arp2/3 interaction and inhibits cell migrationScientific Reports2012ISSN 2045-2322
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Chellaiah, Meenakshi A. et al.Phosphorylation of a Wiscott-Aldrich syndrome protein-associated signal complex is critical in osteoclast bone resorptionJournal of Biological Chemistry2007ISSN 0021-9258
Van Der Gucht, Jasper et al.Stress release drives symmetry breaking for actin-based movementProceedings of the National Academy of Sciences of the United States of America2005ISSN 0027-8424

 

Question 1: By itself, what is the effect of VCA-domain WASP protein on actin polymerization?

Answer 1:  By itself, the VCA-domain WASP protein (Cat. # VCG03) partial inhibits actin polymerization.  When combined with Arp2/3 (Cat. # RP01), actin polymerization is greatly enhanced.  See the associated datasheets for details.

 

Question 2: What is the optimal concentration of Arp2/3 and VCA-domain WASP protein to modify actin polymerization?

Answer 2:  We recommend that pyrene actin is present at a final concentration of 0.8 μM, Arp2/3 complex at 10 nM (Cat. # RP01) and the VCA domain of WASP at 400 nM (Cat. # VCG03). The critical factor here is the actin concentration which must be 0.80 +/-0.2 µM in the final reaction. This concentration is just below the critical concentration for assembly and hence will not polymerize on it’s own.

 

 

If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com