Quantification of individual high molecular weight subunits of wheat glutenin using SDS-PAGE and scanning densitometry
1992
Kolster, P. | Krechting, C.F. | Gelder, W.M.J. van
A method was developed for quantifying the individual high molecular weight (HMW) glutenin subunits present in a wholemeal flour of wheat (Triticum aestivum L.) Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the subunits from each other, as well as from the other proteins present in an extract of flour. The extraction and separation of the HMW glutenin subunits were optimized, which was necessary for accurate quantification of the proteins by scanning densitometry, as was the use of a sensitive method for staining the proteins in the polyacrylamide gels, using a colloidal suspension of Coomassie Brilliant Blue. The stained bands were quantified by scanning densitometry. The coefficient of variation (CV) of the staining intensity of individual HMW glutenin subunits was 12-4% for lanes of the same gel; variability between gels used on the same or different days introduced an additional CV of 7% each. The method developed has several important advantages over the quantification of the subunits by reversed-phase high-performance liquid chromatography (RP HPLC). First, in contrast to RP-HPLC, a crude extract containing all the storage proteins can be used. Second, the proteins can be quantified and identified in the same analysis.
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