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Phase III+: The University is open for expanded research operations; only authorized personnel will be admitted on campus. More info here.
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Phase III+: The University is open for expanded research operations; only authorized personnel will be admitted on campus. More info here.
!
Phase III+: The University is open for expanded research operations; only authorized personnel will be admitted on campus. More info here.

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.