The most reproducible and accurate method of determining the amount of filamentous actin (F-actin) content versus free globular-actin (G-actin) content in a cell population is to use Western blot quantitation of F-actin and G-actin cellular fractions. The general approach is to homogenize cells in F-actin stabilization buffer, followed by centrifugation to separate the F-actin from G-actin pool. The fractions are then separate…
The G-LISA™ assay uses a 96-well plate coated with RBD domain of Rho-family effector proteins. The active GTP-bound form of the Rho-family protein, but not the inactive GDP-bound form, from a biological sample will bind to the plate. Bound active Rho-family protein is then detected by incubation with a specific primary antibody followed by a secondary antibody conjugated to HRP. The signal is then developed with OPD or chemilu…
Cost effective high throughput screen for anti-cancer compounds. Basic research to measure compound IC50s and specificity for tubulin. Screening proteins for effects on tubulin polymerization activity. Teaching aid for undergraduate/graduate class in pharmacology. This assay is an economical one step procedure for determining the effects of drugs or proteins on tubulin polymerization. It is an adaptation of an assay o…
The Actin Polymerization Biochem Kit™ is based on the enhanced fluorescence of pyrene conjugated actin that occurs during polymerization. The enhanced fluorescence that occurs when pyrene G-actin (monomer) forms pyrene F-actin can be measured in a fluorimeter to follow polymerization over time. Also, by using preformed pyrene F-actin, it is possible to follow depolymerization. Both cell/tissue extracts and purified proteins ca…
