Actin Typing on Total Cellular Extracts

Abstract

Based on the finding that the amino-terminal tryptic peptide of actin is a reliable marker for actin divergence, we describe in detail a highly sensitive protein-chemical procedure for actin typing. The method is performed on non-radioactively labelled cells and tissues and six actins can be identified unambiguously in warm-blooded vertebrates. The method is quantitative and gives directly the ratio of the different actins present in the specimens. It does not require previous purification of actin and can be used on total cellular extracts without any prior fractionation. The procedure can be extended to actins not previously characterized by amino acid sequence analysis and makes certain predictions possible about the partial amino acid sequences of the amino-terminal tryptic peptides, mostly sufficient for a correlation with DNA sequences derived from cloned actin genes. This is done as an example for the cytoplasmic actin present in Schneider L-2 Drosophila melanogaster cells. Although the method is currently used routinely on 10⁵ cells, modifications are discussed, which should allow the analysis to be performed with even higher sensitivity.