Sequencing of Protein from a Single Spot of a 2-D Gel Pattern: N-Terminal Sequence of a Major Wheat LMW-Glutenin Subunit

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Abstract
Kasarda, D. D., Tao, H. P., Evans, P. K., Adalsteins, A. E. and Yuen, S. W. 1988. Sequencing of protein from a single spot of a 2-D gel pattern: N-terminal sequence of a major wheat LMW-glutenin subunit—J. exp. Bot. 39: 899–906. Proteins extracted from wheat (Triticum aestivum L.) flour were fractionated by two-dimensional electrophoresis (isoelectric focusing in the first dimension and SDS-polyacrylamide gel electro-phoresis in the second). The proteins in the gel were electroblotted onto a polyvinylidene difluoride membrane and the pattern was developed by staining with amido black. There was considerable variation in the degree of transfer for various proteins; ω-gliadins did not transfer at all, most high-molecular-weight glutenin subunits transferred poorly or not at all, but most other gliadins and some glutenin subunits transferred fairly well. A single spot corresponding to the major low-molecular-weight (LMW) glutenin subunit of the wheat (T. aestivum L.) cultivar ‘Anza’ was cut out and subjected to automatic amino acid sequencing without prior elution from the membrane. The sequence was identical at 12 positions of the 14 cycles to the major sequence reported earlier for a mixture of LMW-glutenin subunits. Yields of phenylthiohydantoin-amino acids ranged from about one to five picomoles for the correctly identified amino acids.

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