A thin-gel isoelectric focusing method for quantitation of protein S-thiolation.
A thin-gel isoelectric focusing method has been developed for analysis of protein S-thiolation (formation of mixed disulfides with low molecular weight thiols). The method is rapid and it can be used with 3 to 5 micrograms of a pure protein, or 15 to 20 micrograms of tissue extract protein. It is possible to detect a modification of the protein sulfhydryl by either charged or uncharged thiols, and to determine the quantity of different S-thiolated protein species in a modified sample. The method was used to quantitate the amount of S-thiolation of phosphorylase b in a reaction with oxidized glutathione that produced four S-thiolated forms of the enzyme. The method was also used to detect S-thiolation of two proteins in a cardiac tissue extract treated with diamide. One of the protein bands was shown to be S-thiolated with both cysteine and glutathione, while the other band was S-thiolated only with glutathione.
D000818:Animals; D002417:Cattle; D007525:Isoelectric Focusing / Q000379:methods*; D009206:Myocardium / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D013438:Sulfhydryl Compounds / Q000032:analysis*
D011506:Proteins; D013438:Sulfhydryl Compounds