Pyrophosphate in the Binding Reaction Increases the Sensitivity of Mobility Shift Analysis

摘要

Gel mobility shift analysis (GMSA), also known as gel retardation, is a fundamental technique used in the study of protein-nucleic acid interactions. Several detection methods available for GMSA can be used, depending on the amount of protein or sensitivity required. These include Coomasie~(R) blue or silver staining, the use of antibodies and autoradiography. We have used GMSA to study the binding between partially purified trypto-phan represser (TrpR) from E. coli to a 241-bp DNA fragment containing the tryptophan operator promoter region. We encountered loss of counts when using partially purified protein during this assay. The problem was solved by the addition of 50 μM py-rophosphate in the incubation reaction.