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Protocol: Reduction and Alkylation Before First Dimension Protocol

The standard method for maintaining reduced cysteines during the first dimension is to add reducing agents, such as DTT or TBP (tributyl phosphine), or disulfide exchange reagents, such as DeStreak, to the rehydration buffer. However, streaking in some samples is significantly reduced if cysteines are both reduced and alkylated before the first dimension using the methods below.

Method for Samples in Solution:

• If the proteins are in a solution with no area or detergents, add urea to a final concentration of 8M. About 80 mg solid urea should be added per 100 ul sample. Samples were allowed to dissolve by vortexing and incubating at room temperature.  Estimate the resulting volume of solution while transferring the sample to a clean microfuge tube. If samples are already in a urea-containing solution, proceed to the next step without modifications.
• Add DTT to a final concentration of 50 mM. This can be can be accomplished by adding 0.77 mg solid DTT per 100 ul sample or by adding 1 part 500 mM DTT to 9 parts solution. Shake samples for 10minutes.
• Add iodoacetamide to each sample for a final concentration of 125 mM.  2.3125 mg dry iodoacetamide should be added per 100 ul sample. Place samples on shaker for 10 minutes.
• Acetone precipitate proteins in the normal manner.