Electrophoretic conditions for high resolution citrus isozymes in polyacrylamide gel electrophoresis.
Electrophoretic conditions including electrode and gel buffers, acrylamide concentration, use of stacking gels, voltage, current, and run time were investigated in order to produce isozyme bands of high resolution which would facilitate densitometric quantification of enzyme activity following polyacrylamide gel electrophoresis (PAGE). Electrode buffers which provided optimal conditions for gels stained for the isozymes of malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (6-PGD), phosphoglucose isomerase (PGI), and shikimate dehydrogenase (SkDH) were 0.02 M Tris-glycine, pH 8.5, 0.1 M sodium borate, pH 6.0, 0.1 M sodium borate, pH 8.7, and 0.07 M sodium borate, pH 7.0, respectively. A 0.5 M Tris-HCl, pH 7.5, gel buffer was optimal for gels stained for the isozymes of 6-PGD, PGI and SkDH. A 0.5 M Tris-HCl, pH 8.5, gel buffer was best for gels stained for MDH. Stacking gels were found to be detrimental to enzyme activity and showed no improvement in resolution for any of the enzymes. Acrylamide concentration for gels stained for MDH were 8.7%, gels stained for 6-PGD and PGI were 7.5%, while gels stained for SkDH had an acrylamide concentration of 5.0%. Higher concentrations above these levels caused a reduction and in some cases loss of band activity, while below this concentration there was a decrease in band resolution.(ABSTRACT TRUNCATED AT 250 WORDS)
King
B J
BJ
University of Central Queensland, Rockhampton.
Lee
L S
LS
Rackemann
R G
RG
Scott
P T
PT
D000429:Alcohol Oxidoreductases / Q000032:analysis / Q000235:genetics; D002021:Buffers; D002957:Citrus / Q000201:enzymology* / Q000235:genetics; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005782:Gels; D005956:Glucose-6-Phosphate Isomerase / Q000032:analysis / Q000235:genetics; D007527:Isoenzymes / Q000032:analysis* / Q000235:genetics; D008291:Malate Dehydrogenase / Q000032:analysis / Q000235:genetics; D010734:Phosphogluconate Dehydrogenase / Q000032:analysis / Q000235:genetics; D018515:Plant Leaves / Q000201:enzymology; D010940:Plant Proteins / Q000032:analysis* / Q000235:genetics
D002021:Buffers; D005782:Gels; D007527:Isoenzymes; D010940:Plant Proteins; D000429:Alcohol Oxidoreductases; C022641:Shikimate dehydrogenase; D008291:Malate Dehydrogenase; D010734:Phosphogluconate Dehydrogenase; D005956:Glucose-6-Phosphate Isomerase