Edgar A J
Department of Biophysics, Cell and Molecular Biology, King's College, London, UK.
Electrophoresis. 1989 Oct;10(10):722-5. doi: 10.1002/elps.1150101012.
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.
使用在含有三磷酸腺苷（ATP）、钙离子（Ca2+）和二硫苏糖醇的缓冲液中预平衡的凝胶和琼脂糖缓冲带，在8 - 25%丙烯酰胺的Pharmacia PhastGels上对单体肌动蛋白（G - 肌动蛋白）进行电泳。在这些凝胶上，相对于标准蛋白质，G - 肌动蛋白以清晰的条带形式迁移，表观分子量为45 kDa，略大于其实际分子量42 kDa。在没有这些溶质的情况下进行电泳会导致蛋白质变性和聚集，表现为一条长长的拖尾。丝状肌动蛋白（F - 肌动蛋白）不进入凝胶。肌动蛋白单体结合蛋白脱氧核糖核酸酶I（DNase I）与G - 肌动蛋白形成二元复合物。通过非变性凝胶电泳确定该复合物的纯度和表观分子量为74 kDa。通过用适当的配体对凝胶和缓冲带进行预平衡这一简单步骤，该技术可扩展用于研究肌动蛋白与其他G - 肌动蛋白结合蛋白以及稳定性依赖于配体的其他蛋白质之间的相互作用。
