Abstract
Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample preparation steps. This modification occurs when iso-cyanate, a urea break-down product, covalently modifies lysine residues, thus inducing a change in isoelectric point. Urea is used at up to 9 M concentrations in sample preparation and 2-D gels because of its ability to disrupt protein structure and effect denaturation without the need for ionic surfactants such as SDS. We have studied carbamylation using 7 M urea and 2 M thiourea, under a range of experimental temperatures to establish when, and if, it occurs and what can be done to minimize the modification. The actual time required for protein extraction from a tissue is usually short compared to the time required for procedures such as reduction and alkylation and IPG rehydration and focusing. Therefore, it is the temperature during these post-extraction procedures that is the most critical factor. Our experiments have shown that carbamylation does not occur during electrophoresis in the presence of urea, even with prolonged run-times. However, under poorly controlled sample preparation and storage conditions, it can become a major event.
Original language | English |
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Pages (from-to) | 239-242 |
Number of pages | 4 |
Journal | Journal of Proteome Research |
Volume | 2 |
Issue number | 3 |
DOIs | |
Publication status | Published - May 2003 |
Keywords
- Alkylation
- Artifacts
- Carbamylation
- MALDITOF mass spectrometry
- Proteomics
- Reduction
- Two-dimensional electrophoresis