Two-dimensional peptide mapping by polyacrylamide-gel electrophoresis with limited proteolysis in SDS |
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Authors: | A Takeda R E Cone |
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Affiliation: | 1. Department of Pathology Yale University School of Medicine New Haven, Connecticut 06510 USA;2. Department of Surgery Yale University School of Medicine New Haven, Connecticut 06510 USA |
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Abstract: | The peptide mapping method described by Cleveland, et al. (1) was improved to a two-dimensional analysis applicable to minute amounts (less than 0.5 microgram) of proteins. Radioiodinated proteins for analysis were purified by electrophoretic elution of the proteins from polyacrylamide gels into buffer containing 0.1% sodium dodecyl sulfate. The proteins were digested enzymatically in the presence of 0.1% sodium dodecyl sulfate and an excess of nonlabeled bovine serum albumin (0.2 mg/ml) relative to labeled substrate in order to attain reproducibility by maintaining a consistent substrate concentration among different samples. The peptides of these limited proteolytic products were resolved by two-dimensional polyacrylamide gel electrophoresis (isoelectric focusing followed by SDS-gels). The resulting 2D-peptide maps of murine and bovine albumin and a murine lymphocyte membrane protein, Tp100, showed excellent resolution and reproducibility. |
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Keywords: | SDS sodium dodecyl sulfate ID- one-dimensional polyacrylamide gel electrophoresis 2D-PAGE two-dimensional polyacrylamide gel electrophoresis IEF isoelectric focusing BSA bovine serum albumin PBS phosphate-buffered saline |
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