Electrophoretic mobility of Alzheimer’s amyloid-β peptides in urea–sodium dodecyl sulfate–polyacrylamide gel electrophoresis
| Autor: | Thomas L. Emmons, John K. Kawooya, Susan C D’Andrea, Patricia A Gonzalez-DeWhitt, Melissa C Camp |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Protein Denaturation
Biophysics Peptide Biochemistry Protein Structure Secondary chemistry.chemical_compound Protein structure Humans Urea Sodium dodecyl sulfate Molecular Biology Protein secondary structure Polyacrylamide gel electrophoresis chemistry.chemical_classification Gel electrophoresis Amyloid beta-Peptides Chemistry Cell Biology Amino acid Electrophoresis Culture Media Conditioned Electrophoresis Polyacrylamide Gel Dimerization Hydrophobic and Hydrophilic Interactions HeLa Cells |
| Zdroj: | Analytical Biochemistry. 323:103-113 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2003.08.027 |
| Popis: | The 43-amino acid Alzheimer's amyloid-beta peptide (Abeta peptide) retains a predominantly alpha-helix and beta-strand structure in sodium dodecyl sulfate (SDS) solution. This conformer has a high tendency to aggregate during conventional SDS-polyacrylamide gel electrophoresis (PAGE). Both the secondary structure and the proclivity for aggregation are obviated by the use of urea-SDS-PAGE: In 8M urea-with or without SDS-the Abeta peptide becomes 100% random coil and remains monomeric. However, during electrophoresis in this medium, the peptide and its truncated variants do not obey the law of mass/mobility relationship that most proteins-including Abeta peptides-follow in conventional SDS-PAGE. Rather, the smaller carboxy-terminally truncated peptides migrate slower than the larger full-length peptide, while the amino terminally truncated peptide does migrate faster than the full-length Abeta peptide. Thus, despite their small size (2-4kDa) and minor differences between their lengths, the Abeta peptides display a wide separation in this low-porosity (12% acrylamide) gel. We found that this unusual electrophoretic mobility in 8M urea is due to the fact that the quantity of [35S]SDS bound to the Abeta peptides, instead of being proportional to the total number of amino acids, is rather proportional to the sum of the hydrophobicity consensus indices of the constituent amino acids. It is then their hydrophobicity and, hence, the net negative charges contributed by the peptide-bound SDS that plays a major role in determining the mobility of Abeta peptides in 8M urea-SDS-PAGE. The high selectivity of the 8M urea-SDS-PAGE method allowed us to detect the presence of hitherto unknown Abeta peptide variants that were secreted in the conditioned medium by cultured HeLa cells. |
| Databáze: | OpenAIRE |
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