The 1990 sequence sample, ABRF-9OSEQ, was designed to be loaded onto a PVDF membrane so that it would simulate a relatively large electroblotted protein on this increasingly popular sequencing substrate. This project provides the first, large scale, systematic study of PVDF sequencing performance. The sample was sent to over 100 member facilities as well as to all ABRF Corporate Sponsors.
More than 50 facilities returned data by early July providing a solid database for analysis of low level sequence performance on PVDF membranes. As anticipated, the average number of residues that could be assigned was far less than in the 1988 and 1989 sequence studies; the accuracy of positive assignments was 83% versus 95% in the two previous sequence studies. These lower values probably reflect both the smaller amount of sample (about 30 pmoles), as well as the fact that it was immobilized on PVDF and had a high background. Analysis of these results is continuing and the good response rate should permit useful comparisons of different sequencing methods such as gas phase vs. Liquid phase cleavage and the importance of glass fiber filters either above or below the PVDF.
As in previous studies, several questions were included on the sequence report form which continue to reveal useful information about protein chemistry facilities. Many features of the average facility remain unchanged from last year while usage of PVDF membranes has increased substantially. In addition, the number of facilities that perform electroblotting in house has increased (69% vs. 53% last year) as well as the percentage of facilities that offer electroblotting as a service (28% vs. 20% last year). The response to a question about the preferred frequency of receiving sequence test samples was especially interesting. This question was included to assess whether yearly distribution of sequence test samples is sufficient or excessive. The vast majority of responding facilities are interested in receiving samples at least once a year, while a substantial number prefer more frequent samples. The high response rate to this year's study and the high level of interest in future samples is especially gratifying.
The sequence of this year's sample and the results of the study will be presented at the ABRF satellite meeting on August 11 and a manuscript will be submitted for publication in the Protein Society Meeting proceedings book, Techniques in Protein Chemistry II which will be edited by J. Villafranca.
The committee has also tabulated the sequence assignments from the 1989 sequence study. Click here to see the tabulated data
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Created: 10th August 1995
Last modified: 21st October 1995