Measurement of protein thiols after heat shock using 3-(-N-maleimido-propionyl) biocytin labeled proteins separated by SDS-PAGE and electroluted onto nitrocellulose: thiol blotting.
We tested the hypothesis that depletion of intracellular glutathione (GSH) during heat shock results in protein thiol oxidation, thereby increasing thermal sensitivity. Depletion of GSH was accomplished using a combination of diethylmaleate and buthionine sulfoximine and protein sulfhydryls were measured using two independent methods. Chinese hamster ovary (CHO) cells were solubilized in polyacrylamide gel electrophoresis (PAGE) sample buffer containing 3-(N-maleimido-propionyl) biocytin, separated by sodium dodecyl sulfate (SDS)-PAGE, electroluted onto nitrocellulose, and visualized via avidin-alkaline phosphatase staining. A second method utilized 5,5'-dithiobis(2-nitrobenzoic acid) to measure protein solubilized in SDS. The results indicate that when CHO cells are heated at 43 degrees C GSH depletion can increase thermal sensitivity but does not cause nonspecific protein thiol oxidation at this temperature or at 37 degrees C.
Freeman
M L
ML
Vanderbilt Center for Radiation Oncology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Meredith
M J
MJ
D000222:Adaptation, Physiological; D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000502:physiology; D006358:Hot Temperature; D007202:Indicators and Reagents; D008239:Lysine / Q000031:analogs & derivatives; D008301:Maleimides; D011506:Proteins / Q000032:analysis*; D013438:Sulfhydryl Compounds / Q000032:analysis*
D007202:Indicators and Reagents; D008301:Maleimides; D011506:Proteins; D013438:Sulfhydryl Compounds; C047272:3-(N-maleimidopropionyl)biocytin; D005978:Glutathione; D008239:Lysine