Reduction & Alkylation
A protein sample is typically reduced & alkylated to break disulfide bridges and ‘cap’ the reduced cysteines. For gel-bands, omission of this step can lead to loss of cysteine-containing peptides because free cysteines can react with acrylamide in the gel which results in that extraction is no longer possible. However, reduction & alkylation requires additional steps and can increase the chance of unintended contamination by for example keratins. For identification-only purposes we recommend to omit reduction & alkylation UNLESS identification of cysteine-containing peptides is needed.
In a report by Anna and Andrej Shevchenko it was shown that for in-gel digestion, the main difference between conducting reductions & alkylation versus omitting this step is the loss of cysteine-containing peptides.
Omitting reduction & alkylation can also speed-up processing and it will lower cost. In the figure below, workflows are shown for an SDS-PAGE protein band digestion w/o and w/ reduction & alkylation.
| Workflow without reduction & alkylation | Workflow with reduction & alkylation |
|---|---|
| Destaining ↓ Dehydration ↓ Addition of trypsin ↓ Removal of excess trypsin ↓ Overnight digestion ↓ Quench digestion |
Destaining ↓ Dehydration ↓ Reduction ↓ Removal of excess reduction agent ↓ Alkylation ↓ Removal of excess alkylation agent ↓ Washing ↓ Dehydration ↓ Addition of trypsin ↓ Removal of excess trypsin ↓ Overnight digestion ↓ Quench digestion |
Workflow for the in-gel digestion with and without reduction and alkylation.