Product Uses Include
Profilin protein has been expressed in E.coli via chromatography without a tag. Thus the protein does not contian a 6xHis or other tag. The protein is supplied as a lyophilized powder. When resuspended in nanopure water, the profilin will be in the following buffer: 10 mM Tris pH 8.0, 1 mM EDTA, 1 mM DTT, 5% (w/v) sucrose and 1% (w/v) dextran.
The lyophilized protein is stable at 4°C desiccated (<10% humidity) for up to 1 year.
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Purity is >95% (see figure 1).
Figure 1. Profilin purity determination. A 10 µg sample of PR02 (profilin molecular weight approx. 15 kDa) was run on an SDS-PAGE gel and stained with coomassie blue. Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat.# ADV02).
Profilin is tested for biological activity by a binding assay with actin. Profilin is bound to poly-L-proline sepharose beads and incubated with G-actin. The profilin beads bind >90% of the available actin by this procedure.
Actin Polymerization Inhibition Assay. The ability of profilin to inhibit actin polymerization was assessed by SDS-PAGE of proportionally loaded supernatant (S) and pellet (P) fractions from G-actin incubated with and without profilin-1 according to the assay method. In the absence of profilin-1, approx. 80% of the actin protein (43kDa) is found in the pellet fraction as F-actin (P, lane 2). When G-actin is incubated with profilin prior to polymerization, only 20% approx. of actin is found as F-actin in the pellet (P, lane 3), while the other 80% remains as G-actin in the supernatant (S, lane 3). Lane 1, profilin-1 protein alone. Mark12 molecular weight markers are from Invitrogen.
Kinetic assay using profilin to inhibit Arp2/3 induced polymerization
Profilin-1 Inhibits Branched Polymerization of Actin Filaments by the Arp2/3 Complex and the VCA Domain of WASP. Actin polymerization was carried out as described in the method; all reactions contain a 1:1 ratio of pyrene- and nonlabeled-actin. Actin in the presence of both Arp2/3 and VCA shows enhancement of actin nucleation. Upon the addition of Profilin-1, the steep nucleation phase provided by both Arp2/3 and VCA is delayed and greatly reduced. The additionof Profilin-1 to actin also decreases the rate of actin polymerization when compared to actin alone.
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|Moseley, James B. et al.||A conserved mechanism for Bni1- and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin||Molecular biology of the cell||2004||ISSN 1059--1524|
|Vignjevic, Danijela et al.||Formation of filopodia-like bundles in vitro from a dendritic network||Journal of Cell Biology||2003||ISSN 0021--9525|
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