Comparative peptide mapping at the nanomole level |
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| Authors: | R.G. Whittaker B.A. Moss |
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| Affiliation: | 1. School of Biochemistry, University of New South Wales, Kensington, New South Wales, 2033, Australia;2. CSIRO, Molecular and Cellular Biology Unit, North Ryde, New South Wales, 2133, Australia |
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| Abstract: | Comparison of highly similar proteins by peptide mapping at the 1-nmol level on cellulose-coated thin-layer sheets is described in detail. Maps were developed in three steps (a) a short chromatographic run in butanol:pyridine:acetic acid:H2O (150:100:3:100), (b) electrophoresis at pH 3.5, and (c) rechromatography in butanol:pyridine:acetic acid:H2O. The inclusion of step (a) greatly reduced streaking in the subsequent electrophoretic direction. Peptides were located by fluorescamine staining and could be eluted for composition or sequence analysis. To ensure complete comparison of the proteins two types of proteolytic digestion, a tryptic and a combined tryptic/thermolytic digestion, were necessary. |
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