Protein Extraction from Cereal Endosperm

Eight endosperms, weighing ~13mg per grain and a total of 100mg, were used for each extraction. Total protein was extracted with 9 vols (w/v) of denaturing buffer containing 0.125M TRIS-HCl, pH 6.8, 8M urea, 4% SDS, and 5% β-mercaptoethanol. Samples were extracted overnight at room temperature, centrifuged at 20 000 g to remove gelatinized starch and other particulate matter, and supernatants were used for SDS–PAGE and western blotting. Soluble proteins were extracted on ice with 9 vols (w/v) (three repeats with 3 vols) of extraction buffer, containing 10mM HEPES-KOH, pH 7.5, 100mM NaCl. After extraction, samples were centrifuged at 20 000 g at 4 °C for 10min. The residual pellet was extracted with 9 vols (w/v) of denaturing buffer as mentioned above and, following centrifugation, the supernatant was used to represent insoluble, starch granule-associated proteins.