A systematic investigation into the recovery of radioactively labeled proteins from sodium dodecyl sulfate-polyacrylamide gels.
We report the results of a systematic investigation designed to optimize a method for quantifying radioactivity in proteins in sodium dodecyl sulfate-polyacrylamide gels. The method involves dissolving appropriately sized pieces of gel in hydrogen peroxide and heating to 70 degrees C overnight followed by liquid scintillation counting. H(2)O(2) had no effect on the count rates of [(14)C]bovine serum albumin (BSA) when counted in a conventional liquid scintillation system, and the count rates remained stable for several days. Temperatures below 70 degrees C resulted in incomplete extraction of radioactivity from gels containing [(14)C]BSA, but there was also a significant reduction in count rates in samples incubated at 80 degrees C. At 70 degrees C recovery was not affected by the amount of sample loaded onto the gel or by the staining procedure (Coomassie Brilliant Blue or SYPRO Ruby). Recoveries were in the range of 89-94%, and the coefficient of variation for five replicate samples was 5-10%. This method offers a reliable way of measuring the amount of radioactivity in proteins that have been separated by electrophoresis. It may be useful, for example, in quantitative metabolic labeling experiments when it is necessary to know precisely how much tracer has been incorporated into a particular protein.
Zhou
Shaobo
S
Nutrition, Food and Health Research Centre, London, UK.
Bailey
Matthew J
MJ
Dunn
Michael J
MJ
Preedy
Victor R
VR
Emery
Peter W
PW
D000818:Animals; D002250:Carbon Radioisotopes; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods* / Q000592:standards; D006861:Hydrogen Peroxide; D007553:Isotope Labeling; D009132:Muscles / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D011868:Radioisotopes; D051381:Rats; D012588:Scintillation Counting; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000302:isolation & purification / Q000592:standards; D013194:Staining and Labeling; D013462:Sulfur Radioisotopes; D013696:Temperature
D002250:Carbon Radioisotopes; D011506:Proteins; D011868:Radioisotopes; D013462:Sulfur Radioisotopes; D012710:Serum Albumin, Bovine; D006861:Hydrogen Peroxide