| Popis: |
We have quantitatively examined the various parameters affecting the electrotransfer and sequence analysis of proteins from sodium dodecyl sulfate (SDS) gels to derivatized glass fiber paper or to polyvinyldifluoride (PVDF) membranes. Transfer yields in the range of 90–95% can be obtained for proteins in the molecular weight range of 10–90 kDa for transfer from 12% SDS gels to glass fiber paper derivatized with either QAPS (N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride) or APS (aminopropyltriethoxysilane). In order to achieve these yields, it was necessary to modify the conditions described by R. Aebersold et al. (J. Biol. Chem.261, 4229–4238, 1986). We activated the glass fiber paper with dilute ammonia water and derivatized the activated glass fiber paper with OAPS and APS in anhydrous solvents which were allowed to slowly absorb moisture during the derivatization process. The transfer yield varied with transfer time versus molecular weight of the protein for a given percentage gel. Shorter transfer times and higher yields were obtained for higher molecular weight proteins on 8% gels. Lower molecular weight protein gave higher yields from 12% gels under similar transfer conditions. Sequencing yields of the transferred proteins were in the range of 10–80%, but a number of background peaks were observed on HPLC analysis of the phenylthiohydantoin amino acid derivatives. Transfer yields in the range of 85–95% were observed for similar experiments with PVDF membranes. In order to achieve these yields, it was necessary to modify the conditions described by P. Matsudaira (J. Biol. Chem.262, 10035–10038, 1987). A lower voltage and longer transfer times gave higher transfer yields. In order to achieve consistently high transfer yields, it was also necessary to precoat the PVDF membranes with Polybrene. The PVDF membranes were cut into approximately 1-mm-wide strips and inserted into a continuous flow reactor (J. E. Shively, P. Miller, and M. Ronk, Anal. Biochem.163, 517–525, 1987) for sequence analysis. Overall yields of samples loaded onto gels, electrotransferred to Polybrenecoated PVDF membranes, and sequenced ranged from 50–60% for β-lactoglobin (10–50 pmol loaded onto SDS gels) to 20–30% for bovine serum albumin and soybean trypsin inhibitor (50 pmol loaded onto SDS gels). A comparison of the two methods shows clear advantages for the PVDF membranes over the derivatized glass fiber paper, including the ability the ability to directly sequence the Coomassie blue-stained PVDF membranes, and the lower backgrounds observed on subsequent sequence analysis. |
