Disulfide bond reduction using the ReadyPrep reduction-alkylation kit. Protein samples were reduced either
using 50 mM DTT in rehydration/sample buffer or by treatment with the reduction-alkylation kit.
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.
Disulfide bond reduction ensures proper protein migration and often yields better resolution of protein spots, fewer streaks, and greater reproducibility in 2-D separations.
- Is essential for 2-D analysis of basic proteins
- Permanently eliminates disulfide bonds prior to IEF
- Reduces proteins using tributylphosphine (TBP), then alkylates proteins with iodoacetamide
