IN-GEL CYSTEINE REDUCTION AND ALKYLATION

Solutions:

• 10 mM DTT/100 mM NH4HCO3 (15.4 mg of DTT in 10 ml of 100 mM NH4HCO3)
  
  
• 55 mM iodoacetamide/ 100 mM NH4HCO3 (0.102 g of iodoacetamide in 10 ml of 100 mM NH4HCO3
  

Procedure:

1. After step 2 of the the in gel tryptic digestion protocol, add sufficient 10 mM DTT/100 mM NH4HCO3 to cover the gel pieces and incubate for 45 minutes at 56°C.
   
   
2. Cool tubes to room temp. Remove excess liquid, and quickly replace with about the same volume of freshly prepared 55 mM iodoacetamide/ 100 mM NH4HCO3 solution.
   
   
3. Incubate for 30 minutes at room temp in the dark.
   
   
4. Remove iodoacetamide solution, and continue with step 3 in the tryptic digestion.