ReadyPrep™ Reduction-Alkylation Kit



Reduction and Alkylation

Disulfide bond removal using the ReadyPrep™ reduction-alkylation kit. Protein samples were reduced using either 50 mM DTT in rehydration/sample buffer (before) or the reduction-alkylation kit (after). Both samples were applied by cup loading onto 11 cm ReadyStrip™ pH 7–10 IPG strips and focused for the first dimension, then run on Criterion™ 8–16% Tris-HCl precast gels for the second dimension.

The ReadyPrep reduction-alkylation kit reduces and irreversibly blocks the formation of inter- and intramolecular disulfide bonds prior to isoelectric focusing (IEF). Disulfide bond formation is particularly problematic for basic proteins because of the increased rate of their formation in an alkaline environment. Moreover, many reducing agents become negatively charged during IEF and migrate off the IPG strip, thus allowing disulfide bonds to re-form (Görg 2000, Hoving 2002).

The ReadyPrep reduction-alkylation kit:

  • Eliminates the potential for disulfide bond formation during IEF
  • Reduces disulfide bonds with tributylphosphine (TBP) and alkylates the sulfhydryl groups with iodoacetamide (Herbert et al. 2001)
  • Provides reagents for treatment of 100 samples (300 µl each, containing up to 10 mg/ml of protein)
  • Performs treatment directly in the rehydration/sample buffer, in less than 2 hours, and with very little hands-on time
  • Is compatible with other ReadyPrep sample preparation kits and buffers

Görg A et al. (2000). The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 1,037–1,053.
Herbert B et al. (2001). Reduction and alkylation of proteins in preparation of two-dimensional map analysis: Why, when and how? Electrophoresis 22, 2,046–2,057.
Hoving S et al. (2002). Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients. Proteomics 2, 127.

ReadyPrep™ Reduction-Alkylation Kit

100 preps, general purpose protein preparation kit for reducing gel streaking and improving resolution of basic proteins

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4110063 Instruction Manual, ReadyPrep Reduction-Alkylation Kit, Rev A Click to download