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-32P]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 inorganic phosphate. To follow the kinetics of the CheA-CheY phospho-transfer reaction and the subsequent dephosphorylation of phospho-CheY, we separated the reaction components by polyacrylamide gel electrophoresis and measured the amount of 32P label in the CheA. CheY and inorganic phosphate bands with phosphor storage screens. By reducing the time 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 much larger proteins. In principle, any radiolabeled molecules that can be separated by relatively rapid means, such as acrylamide gel electrophoresis, and that are detectable with a phosphor storage screen, should be amenable to this technique.
University of Utah, Salt Lake City.
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Morrison
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Parkinson
D000255:Adenosine Triphosphate; D001426:Bacterial Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D000071677:Histidine Kinase; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D000072236:Methyl-Accepting Chemotaxis Proteins; D010710:Phosphates / Q000032:analysis; D010761:Phosphorus Radioisotopes / Q000032:analysis*; D010766:Phosphorylation; D010957:Plasmids; D011494:Protein Kinases / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D010710:Phosphates; D010761:Phosphorus Radioisotopes; C000607606:cheY protein, E coli; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000071677:Histidine Kinase; C000607608:cheA protein, E coli