Detection of actin-binding proteins in human platelets by 125I-actin overlay of polyacrylamide gels.

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Abstract
Actin-binding proteins were identified in human platelets with a gel-overlay technique that uses 125I-G-actin. Platelet proteins were separated on SDS [sodium dodecylsulfate] polyacrylamide gels using the buffer system of Laemmli. The proteins were fixed in the gels with methanol-acetic acid, the SDS was washed out and the proteins were renatured. The gels were incubated with 125I-G-actin from rabbit skeletal muscle that was radiolabeled with 125I. They retained biological activity. After nonspecifically bound radioactivity was washed out, gels were dried and processed for autoradiography. The 125I-G-actin binds to several proteins in human platelets, platelet extracts and the particulate fraction. Control experiments, demonstrate that the 125I-G-actin can be displaced by use of increasing amounts of unlabeled actin, that the binding is stable to 0.6 M NaCl and that preheating the 125I-G-actin to 90.degree. C for 3 min eliminates all binding. Prominent 125I-G-actin-binding activities were present at MW 90,000 and 40,000. The binding to the 90,000 MW protein appears to be at least partially Ca2+ sensitive. The binding to the 40,000 MW protein does not. 125I-G-actin bound to proteins in the SDS gels can be fixed in situ and compared directly with the stained gel. This technique should prove generally useful in identification and purification of some actin-binding proteins from cells and tissues.