Reducing agents are commonly used during sample preparation to cleave disulfide bond crosslinks within proteins and between protein subunits. Disulfide bond formation is particularly problematic for basic proteins because of the increased rate of their formation in an alkaline environment. Disulfide bond formation limits protein solubility, which can interfere with resolution.
Bio-Rad offers the following reducing and alkylating reagents:
- Dithiothreitol (DTT), a commonly used general sulfhydryl reducing agent. DTT has a pI of about 8 and may not remain effective throughout IEF. It may become negatively charged during IEF and migrate off the IPG strip, thus allowing disulfide bonds to re-form (Görg 2000, Hoving 2002)
- Tributylphosphine (TBP), a phosphine reductant that can be used at lower concentrations than DTT and is active over a wider pH range. TBP remains with proteins throughout focusing
- Iodoacetamide, an alkylating agent that can be used following treatment with a reducing agent to permanently modify protein sulfhydryls, preventing proteins from aggregating and precipitating due to oxidative crosslinking. This ensures that proteins remain soluble throughout electrophoresis and produces a spot pattern with more spots, fewer streaks, and greater reproducibility, particularly in the basic regions of 2-D gels
TBP and iodoacetamide are provided as components of the ReadyPrep reduction-alkylation kit, which also provides a complete protocol for performing irreversible reduction and alkylation reactions.