Product Uses Include
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 is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are >95% pure (Figure 1).
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).
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.
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.
|Bogucka-Janczi, Katarzyna et al.||ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization.||eLife||2023||ISSN 2050--084X|
|Bock, Fabian et al.||Rac1 promotes kidney collecting duct integrity by limiting actomyosin activity||Journal of Cell Biology||2021||ISSN 1540-8140|
|Chen, Yuewen et al.||Coronin 2B regulates dendrite outgrowth by modulating actin dynamics||FEBS Letters||2020||ISSN 1873-3468|
|Lin, Shan Shan et al.||Dynamin-2 Regulates Postsynaptic Cytoskeleton Organization and Neuromuscular Junction Development||Cell Reports||2020||ISSN 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 invasion||Molecular Cancer Research||2019||ISSN 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 force||PLoS ONE||2019||ISSN 1932-6203|
|Mikhaylova, Marina et al.||Caldendrin Directly Couples Postsynaptic Calcium Signals to Actin Remodeling in Dendritic Spines||Neuron||2018||ISSN 1097-4199|
|Tu, Ye et al.||EFhd2/swiprosin-1 regulates LPS-induced macrophage recruitment via enhancing actin polymerization and cell migration||International Immunopharmacology||2018||ISSN 1878-1705|
|Schaffer, Ashleigh E. et al.||Biallelic loss of human CTNNA2, encoding αN-catenin, leads to ARP2/3 complex overactivity and disordered cortical neuronal migration||Nature Genetics||2018||ISSN 1546-1718|
|Zhao, Miao et al.||Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization||Oncotarget||2017||ISSN 1949-2553|
|Rondina, M. T. et al.||Non-genomic activities of retinoic acid receptor alpha control actin cytoskeletal events in human platelets||Journal of Thrombosis and Haemostasis||2016||ISSN 1538-7836|
|San Ruiz-Miguel, José E. et al.||The role of Arp2/3 in growth cone actin dynamics and guidance is substrate dependent||Journal of Neuroscience||2014||ISSN 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 actin||Cancer Prevention Research||2014||ISSN 1940-6215|
|Xavier, Charles Peter et al.||Phosphorylation of CRN2 by CK2 regulates F-actin and Arp2/3 interaction and inhibits cell migration||Scientific Reports||2012||ISSN 2045-2322|
|Rao, Mala V. et al.||The myosin Va head domain binds to the neurofilament-L rod and modulates endoplasmic reticulum (ER) content and distribution within axons||PLoS ONE||2011||ISSN 1932-6203|
|Chellaiah, Meenakshi A. et al.||Phosphorylation of a Wiscott-Aldrich syndrome protein-associated signal complex is critical in osteoclast bone resorption||Journal of Biological Chemistry||2007||ISSN 0021-9258|
|Van Der Gucht, Jasper et al.||Stress release drives symmetry breaking for actin-based movement||Proceedings of the National Academy of Sciences of the United States of America||2005||ISSN 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.
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