Using a reconstituted complex of profilin and skeletal muscle actin as an antigen, we generated a monoclonal mouse antibody against actin, termed 2G2. As revealed by immunoblots of proteolytic actin fragments and by pepscan

Actin is one of the most abundant proteins in eucaryotic cells and is highly conserved during evolution (Hightower and Meagher, 1986). Since the actin isoforms of one organism (Herman, 1993) as well as the actins of different organisms differ only in a relatively small number of amino acids (for a review see Sheterline and Sparrow, 1994), the generation of actin antibodies has been notoriously difficult. Numerous attempts have been made to overcome the low immunogenicity of actin, and to obtain actin antibodies with high affinity, for example, by raising antibodies against SDS-denatured actin (Lazarides and Weber, 1974), against synthetic peptides derived from the actin sequence (Skalli et al., 1988; Gimona et al., 1994) and against complexes of actin with an actin-binding protein (Polzar et al., 1989). Moreover, even after mastering these difficulties, the antiactins generated may be of limited applicability. Since in vivo actin exists in different states (filamentous actin, non-filamentous actin or as a complex with actin binding proteins) the detection of actin with antibodies may be selective. In addition, actin is only one member of a large superfamily of proteins which also comprises actin-related proteins (arps) with highly homologous sequence regions (Fyrberg et al., 1994; Schroer et al., 1994; Mullins et al., 1996), and actin antibodies may therefore recognise actin-related proteins as well. With the exception of those antibodies which were generated against specific peptides, the locations of antiactin epitopes are unknown. Therefore, the interest in epitopemapped anti-actins of high affinity is scientifically justified. For example, they are more versatile in recognising different states and suprastructures of actin, as compared to the actin binding drug phalloidin which is restricted to F-actin binding. Thus, anti-actins have identified actin in the nucleus in addition to cytoplasmic actin. 797 Journal of Cell Science 112, 797-809 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS3798