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Combination of line immunoelectrophoresis and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for molecular weight evaluation of isozymes: Application to iso‐α‐amylases identified in barley and rice seeds

A procedure for enzyme purification by means of line immunoelectrophoresis compatible with subsequent sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the purified enzyme and Coomassie Blue staining is described. Electrophoresis was carried out in an agarose gel backed to GelBond film. The enzyme activity was detected after immunoelectrophoresis on the wet gel and the immunoprecipitate by protein staining on the dried gel. The strip bearing the maximum amount of precipitate was excised and the dried agarose gel scratched off the GelBond support. The dried gel was extracted with the Laemmli sample buffer, heated at 100 °C for 10 min and submitted to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Proteins were stained with Coomassie Blue. Difficulties due to the presence of the immunoglobulin G subunits were encountered. The validity of the procedure was checked with an α‐amylase from barley purified by immunoaffinity chromatography. The procedure was used to compare the molecular weight of α‐amylase isozymes identified in barley and in rice seeds. In barley, α‐amylase I, the minor enzyme group appearing during germination, had a slightly higher molecular weight than α‐amylase II, the main enzyme group. In germinating rice seeds, the minor enzyme group, antigen D, was found to have a molecular weight slightly lower than the one of the other rice α‐amylases. A new α‐amylase antigen identified in rice as the constituent with the lowest pI was called pre‐A. Its molecular weight was found to be the same as the one of the main enzyme group, antigen (A + B).