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Identification of rat liver glutathione S ‐transferase Yb subunits by partial N ‐terminal sequencing after electroblotting of proteins onto a polyvinylidene difluoride membrane from an analytical isoelectric focusing gel
Author(s) -
Chang LiHsueh,
Hsieh JyhChang,
Chen WoanLing,
Tam Ming F.
Publication year - 1990
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150110710
Subject(s) - electroblotting , isoelectric focusing , chromatography , chemistry , isoelectric point , gel electrophoresis , sodium dodecyl sulfate , polyacrylamide gel electrophoresis , coomassie brilliant blue , glutathione , silver stain , biochemistry , staining , microbiology and biotechnology , biology , enzyme , genetics
Abstract Rat liver glutathione S ‐transferases were partially purified using S ‐hexyl glutathione affinity chromatography, followed by native isoelectric focusing employing a pH 7–11 or pH 3–10 gradient. Proteins were excised and eluted from the gel for determination of subunit composition using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. In separate experiments, isoelectric focusing gels were equilibrated with a sodium dodecyl sulfate‐containing buffer at high pH, and proteins on the gel were electroblotted onto a polyvinylidene difluoride membrane, utilizing graphite plates as electrodes. The membrane‐bound proteins were visualized by Coomassie Brilliant Blue staining. The protein bands were then excised from the membrane and inserted into a gas phase sequenator for direct sequencing. N ‐Terminal sequences thus determined were compared with published cDNA sequences. The isoelectric points (p I s) and positions on the isoelectric focusing gel of Yb 1 Yb 1 , Yb 1 Yb 2 and Yb 2 Yb 2 subunits were determined. We have also located on the pH 3–10 focusing gel an N ‐terminal blocked glutathione S‐transferase which has a molecular weight similar to Yb subunits.