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. Gels stained for MDH yielded best results when run for 6.5 h at a constant current of 5 mA/gel and an initial voltage of 40 V, gels stained for 6‐PGD were best after 10 h at an initial current of 8 mA/gel and a constant voltage of 140 V, gels stained for PGI were run for 22 h at an initial current of 9 mA/gel and a constant voltage of 34 V and gels stained for SkDH were run for 10 h at an initial current of 5 mA/gel and a constant voltage of 60 V. Triscitrate buffers used widely in Citrus and other taxons on both starch and polyacrylamide gels were found to be unsatisfactory. Higher molarity buffers with lower current and longer run times were found to provide superior resolution and band separation in comparison to lower molarity buffers with higher current and shorter run times. Zones of activity previously reported in Citrus but not in mandarin cultivars were revealed for both MDH and PGI. Our interpretation of the alleles for SkDH and 6‐PGD were not in agreement with those previously reported for the cultivars studied. These electrophoretic conditions provide isozyme bands of high resolution on PAGE, which will be suitable for densitometric analysis.
- Enzyme visualization
- Polyacrylamide gel electrophoresis