Choose the ReadyPrep Reduction-Alkylation Kit to reduce disulfide bonds and block their formation in isoelectric focusing (IEF) samples. The kit contains tributylphosphine (TBP) to reduce both intramolecular and intermolecular disulfide bonds, and iodoacetamide to then alkylate the thiols.
- Improve IEF resolution and reproducibility
- Eliminate disulfide bond formation during IEF
- Obtain a consistent spot pattern, and minimize streaking and aberrant spot migration
- Use directly in rehydration/sample buffer; complete in less than 2 hours, with very little hands-on time
- Compatible with other ReadyPrep sample preparation kits and buffers
The ReadyPrep Reduction-Alkylation Kit is a simple and effective method to improve the results of 2-D electrophoresis. Many reducing agents become negatively charged during isoelectric focusing and migrate off the IPG strip. Furthermore, dithiothreitol (DTT), the most commonly used reducing agent, is not effective under acidic conditions. TBP has been shown to be more effective reducing agent than DTT for 2-D gel electrophoresis. Additionally, alkylation improves the resolution of basic proteins, which have an increased rate of disulfide bond formation in an alkaline environment (Görg et al. 2000, Hoving et al. 2002).
The combination of reduction with TBP and the subsequent alkylation of the thiols with iodoacetamide is effective in reducing intramolecular and intermolecular disulfide bonds and preventing the reformation of disulfide bonds and disulfide shuffling, resulting in higher resolution, less spot ambiguity, and increased reproducibility.
For treatment of 100 samples, each 300 µl containing up to 10 mg/ml protein
- TBP (200 mM), 0.6 ml, 3 ampoules
- Empty glass vials for TBP storage after opening the ampoules, 3
- Iodoacetamide, 56 mg, 5 amber vials
- Alkylation buffer, 2 ml
- Instruction manual
Görg A et al. (2000). The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 1037–1053. PMID: 10786879
Hoving S et al. (2002). Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients. Proteomics 2, 127. PMID: 11840558