This year has been quite exciting for our committee. Thanks to all of you, we achieved our goal of completing the annual study earlier than usual and with a record number of 77 participants. The data were presented at the Protein Society Symposia in Davos and Boston as well as at the ABRF meeting in Boston. We have prepared a manuscript for publication in Techniques in Protein Chemistry VII. A summary of this study is below.
The ABRF-95AAA study revisited the analysis of protein bound to PVDF-membrane, which was last examined with the ABRF-90AAA3 sample. Ample protein (10 ug/ membrane) was provided in triplicate so that sensitivity was not a limiting factor. Pieces of membrane without bound protein but otherwise treated identically were also included in triplicate to assess the utility of this control. Study participants were not asked to provide information on synthesis costs.
The number of participants (77 sites) surpassed any previous study providing us with 215 sample and 217 blank analyses. The pre-column (52%) and post-column derivitization methods were almost equally used for analyses of the ABRF-95AAA samples. Ninhydrin and PITC were still overwhelmingly the preferred methods.
The results of these PVDF analyses were remarkable for their variability. The overall average yield +/- standard deviation for the sample was 220 +/- 102 pmol (4.1 +/- 1.9 ug). The range of yields was 21-560 pmol (0.4-11 ug protein). There was no significant difference in yield for the pre- or post-column methods or for the additives used. The accuracy of this study was improved over the ABRF-90AAA3 sample (21.4 vs. 26.9%). However, this is about twice the error compared to the ABRF-90AAA1 (10.9%) and ABRF-94AAA1 (13.5%) samples, which were soluble proteins.
All chemistries used detected high amounts of amino acids (0.037-9.45 ug) on blank membranes. The type of additive did not appear to affect the background levels detected. This high background was surprising and would certainly compromise the quality of analysis.
In conclusion, there was some improvement in amino acid composition analysis of PVDF-membrane-bound proteins, but the difference between the various methodologies used was insignificant and the error was nearly two-fold higher than observed for soluble proteins. Perhaps of greatest concern to core facilities is the very high error associated with membrane-bound samples. The unexpectedly high background of free amino acids, shown with the blank membrane analyses, appears to be the cause of the high errors observed. Further work is required to document that possibility because these protein samples were merely adsorbed, rather than electroblotted, onto membrane. Nevertheless, core facility personnel should be aware of the potential for very high errors in this type of analysis.
We are already testing the ABRF-96AAA sample, which focuses on quantitative amino acid analysis and identification of proteins from their composition. We hope to present the results at the ABRF '96: Biomolecular Techniques meeting at San Francisco in March, 1996.
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