Heating of Freeze-dried Protein Samples with Urea for SDS-PAGE in Proteomies Study

Though urea helps solubilize the protein samples, use of urea under the boiling condition is generally avoided due to carbamylation of protein by cyanate in equilibrium with urea. Validity of adding urea in standard sample buffer and subjecting the standard protein, bovine serum albumin, to SDS-PAGE according to Laemmli procedure, which invariably involves heating of the protein sample at 100 degrees C, was evaluated. Heating samples with crystalline ultra-pure urea improved separation, and the peptide mass fingerprint of the gel-separated protein by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometer (MALDI TOF/TOF MS) indicated that the protein could be identified without difficulty with 39.2% of peptide coverage, which was comparable to the value obtained from the protein not treated with urea.