Gel electrophoresis of native actin and the actin-deoxyribonuclease I complex

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Abstract
Electrophoresis of monomeric actin (G‐actin) on 8–25 % acrylamide Pharmacia PhastGels was carried out using gels and agarose buffer strips preequilibrated in buffer containing adenosine triphosphate (ATP), calcium ions (Ca2+) and dithiothreitol. On these gels G‐actin ran as a sharp band at an apparent molecular mass of 45 kDa relative to standard proteins which is slightly greater than its actual molecular mass of 42 kDa. Electrophoresis in the absence of these solutes led to denaturation and aggregation of the protein, as reflected by a long streak. Filamentous actin (F‐actin) did not enter the gel. The actin monomer‐binding protein, deoxyribonuclease I, (DNase I) forms a binary complex with G‐actin. The purity and apparent molecular mass 74 kDa of this complex were determined by native gel electrophoresis. By the simple procedure of preequilibrating both gel and buffer strips with appropriate ligands, this technique could be extended to investigate interactions between actin and other G‐actin‐binding proteins and other proteins whose stability is ligand dependent.