Detection of β-tubulin in tomato seeds:: Optimization of extraction and immunodetection

SDS-PAGE profiles of SDS-denaturated total protein (MODIL) extracts of imbibing tomato seeds showed no large qualitative differences from those of water-soluble (HEPES) proteins. However, the protein concentrations of the HEPES samples were significantly lower than the MODIL ones. The presence of SDS and DTT in MODIL buffer at an alkaline pH were essential for high yields of beta-tubulin. The absence of DTT in HEPES extraction buffer may have allowed oxidation and subsequent loss of beta-tubulin extracted from root tips of germinated seeds. Optimization of the beta-tubulin extraction procedure and improvements of the immunodetection system enabled us to demonstrate the beta-tubulin accumulation pattern in whole tomato seeds and in the different tissues. beta-Tubulin accumulation in seeds was shown to be tissue-dependent, the highest concentration being found in embryo root tip tissue. (C) 1998 Elsevier Science Ltd. All rights reserved.