Bio-Rad ReadyPrep™ Reduction-Alkylation Kit - Manual (Page 8)

For best results, the protein concentration of the
sample should be determined. We recommend the
Bio-Rad RC DC
™ Protein Assay for protein quatitation
because the assay is suitable for the widest variety of
samples and works in the presence of detergents,
reducing agents, and other agents that interfere with
other protein assays.
Note: The protein sample must be free of reducing agent
(i.e., TBP, DTT, or TCEP) before the reduction-alkylation
reaction is performed. This ensures the necessary molar
excess of iodoacetamide to reducing agent during the
reaction. Additionally, the sample must have a low salt
concentration (<40 mM) after the reduction-alkylation
reaction is performed to maintain low conductivity during
IEF. The reduction-alkylation reaction itself contributes salt
to the sample (30 mM). Therefore, it may be necessary to
remove excess salt prior to IEF. (Alternatively, the
reduction-alkylation reaction can be performed in a higher
protein and salt concentration and subsequently diluted for
IEF.) The ReadyPrep 2-D cleanup kit (catalog #163-2130)
can be used to remove excess reducing agent, salt, and
detergent from the sample, and to concentrate the protein
in the sample, if desired.
3. Adjust the pH of the sample to between 8.0 and 9.0
by adding 15 µl of the alkylation buffer. Mix well by
vortexing.
4. Add 12.5 µl of TBP for a final concentration of 5 mM.
Mix well by vortexing. Incubate at room temperature
for 30 min.
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