Urea extraction buffer

9 M urea

4% CHAPS

0.5% IPG buffer (pH 3-10) (AP Biotech)

50 mM DTT

H₂O

Dissolve 5.4 g of urea, 0.4 g of CHAPS, 50.0 μl of IPG buffer, and 0.077 g of DTT in H₂O to a final volume of 10 ml.

If the bicinchoninic acid (BCA) assay will be used to quantitate proteins after extraction, omit DTT from the urea extraction buffer because DTT interferes with the assay. After samples are assayed, add DTT to the sample to a final concentration of 50 mM.