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.