To achieve the maximum of 100 assays given the volumes of cytochalasin and phalloidin provided, the researcher can only use 100 -200 ul of the total lysate sample for the centrifugation to separate G and F-actin. Thus, while a larger volume (1 ml) of lysis buffer can be used, a smaller aliquot (100-200 ul) should be used for the actual assay. If the supply of included cytochalasin runs out, an alternative depolymerization buffer that can be used is 8M urea, 15mM betamercaptoethanol and 10 mM Tris pH 8.0 can be used to denature the F-actin. We also did not include enough cytochalasin or phalloidin for every experimental sample. The expectation is that experimental samples will be grouped together and 1 positive and 1 negative control sample will be run alongside a large group of experimental samples.
The non-muscle actin polymerization assay is performed by using a mixture of pyrene-labeled muscle actin (cat# AP05 or actin polymerization kit, cat# BK003) plus a majority of unlabeled non-muscle actin (cat# APHL99). The pyrene muscle actin will not polymerize efficiently on its own at the concentration used in this assay, so the reaction is dependent on non-muscle actin polymerization for F-actin formation. Pyrene non-muscle actin has been shown to be unstable at the normal storage conditions and so was discontinued. Please click here to download a polymerization assay manual using non-muscle actin.