Two-dimensional gel electrophoresis for identifying proteins that bind DNA or RNA.

摘要: Electrophoretic mobility shift assays (EMSAs) are commonly used to analyze nucleic acid-protein interactions. When acid is bound by protein, its during gel electrophoresis reduced. Similarly, the final position of protein within a complex shifted when compared free state. Here we provide protocol for simple approach that uses these differences identify acid-binding proteins. Following EMSA, denaturing implemented second dimension separation. Protein binds specific identified as spot(s) whose presence at particular position(s) dependent on initial binding reaction. The polypeptide in spot can be subsequently mass spectrometry. As EMSAs performed using partially purified or cell extracts, this substantially reduces need purification. It should facilitate identification approximately 4 d.