Methodology Studies Of Proteomics And The Analysis Of The Prteomic Map Of The Rice Anther Separated By 2D-Page

proteome is the total protein complement of a genome. Proteomic methodology includes techniques of isolation and characterization of all the expressed proteins in cell. 2-D gel electrophoresis for protein isolation and the identification of the protein spots are the core techniques.Using the crude venom of Selenocosmia huwena, finer 2-D electrophoresis maps were gained by optimizing crucial factors and procedures such as sample treatment, electrophoresis parameter, and gel concentration. N-terminal microsequencing and peptide mass fingerprint analysis were used to identify the protein spot on 2D gel. Venom proteins of the spider Selenocosmia huwena were separated by two-dimensional gel electophoresis With the separation in the first dimension on a wide range of immobilized pll (3-10) gradients. Over 300protein spots were presented on a silver-stained 2D gel. The protein spots- with molecular weight > 10 kDa were analyzed, after electrotransferring to polyvinyldene dif luoride (PVDF) membrane, by N-terminal microsequencing. Some of the silver-stained protein spots with molecular weight over 10 kDa were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including (1) in-gel reduction, alkylation, and enzymatic digestion; (2) extracted and desalting by using the pipette tip containing a smallCIS microcolunm(ZiptipTM); and (3) direct MADLI-TOF mass analysis and protein database searching. Several known toxin such as HWTX-I, HWTX-II, HWTX-IV, and SHL-I were identified and some new components were found among these protein spots.The proteins of thermo-sensitive genetic sterile rice anther were analyzed with the methods that we have established. A mean standard deviation of about 1. 45+0. 23mm for IPG-IEF, 1. 15+0. 17mm for SDS-PAGE of position reproducibi1ity has been obtained. The values of isoelectric point (pI) and molecular mass of proteins were obtained by using computer software PDQUEST. Over 1000 spots were obtained on each map. More than 20 protein spots of sterile rice anther on gels were finally identified by MADLI-TOF-MS.