QUANTIFYING RADIOLABELED MACROMOLECULES AND SMALL MOLECULES ON A SINGLE GEL

A protein phosphorylation cascade involved in chemotactic signaling in Escherichia coli was investigated with purified components in vitro. CheA, an auto-phosphorylating histidine kinase, was mixed with [gamma- P-32]ATP, and the labeled protein was purified for use as a reagent in the assays. CheY a response regulator protein, can acquire phosphate groups from CheA but then undergoes rapid hydrolysis, which releases i norganic phosphate. To follow the kinetics of the CheA-CheY phospho-tr ansfer reaction and the subsequent dephosphorylation of phospho-CheY, we separated the reaction components by polyaclylamide gel electrophor esis and measured the amount of P-32 label in the CheA, CheY and inorg anic phosphate bands with phosphor storage screens. By reducing the ti me needed to separate and quantify the reaction products, we minimized diffusive spreading of the low molecular weight inorganic phosphate, which enabled us to measure it accurately on the same gel with the muc h larger proteins. In principle, any radiolabeled molecules that can b e separated by relatively rapid means, such as acrylamide gel electrop horesis, and that are detectable with a phosphor storage screen, shoul d be amenable to this technique.