The Wx protein and the four other minor starch bound proteins are firmly held in the starch granule by a combination of electrostatic forces and entrapment within the starch matrix. The proteins could not be extracted from intact granules without first swelling the starch granules past the gellation point by either heating or treatment with 8M urea. Once the matrix is opened the following conditions have been found to be effective for solubilizing the proteins: 2% SDS, 8M urea (swelling and dissociation are one step), and low molarity buffer (0.01 M) below pH 2 or above pH 7.5. pH extraction was not successful with 0.05M buffers. Extractions at pH 7.5 and above also required a reducing reagent such as 5% BME or 0.01M dithiothreitol. The efficiency of extraction increased with pH from 7.5 to 10.0. pH 2 is as efficient as pH 10 and both are nearly as efficient as SDS or urea.
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