Degradation artefacts during sample preparation for sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Preparation of samples for sodium dodecyl sulphate polyacrylamide gel electrophoresis routinely involves heating the protein in solution containing detergent and reducing agent for at least two minutes. Here we show that this treatment causes fragmentation of the protein glycogen phosphorylase, whether purified or as a component of a skeletal muscle preparation. The fragments are detected as minor bands on western blots and represent the products of discrete breakage point in the peptide sequence. Protease inhibitors cannot suppress the fragmentation. Such small amounts of immunoreactive fragments may be incorrectly identified on western blots as contaminants that were originally present in the antigen preparation. They may also be a source of ambiguity in studies that search for degradation intermediates during proteolysis.
Cookson
E J
EJ
Department of Biochemistry, University of Liverpool.
Beynon
R J
RJ
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006358:Hot Temperature; D007124:Immunoenzyme Techniques; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments; D006005:Phosphorylases / Q000276:immunology / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate; D013048:Specimen Handling / Q000379:methods*
D010446:Peptide Fragments; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D006005:Phosphorylases