Abstract
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°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.
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References
Bennion BJ and Daggett V (2003) The molecular basis for the chemical denaturation of proteins by urea. Proc Natl Acad Sci USA 100, 5142–5147.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.
Cañas B, Piñeiro C, Calvo E, López-Ferrer D, and Gallardo JM (2007) Trends in sample preparation for classical and second generation proteomics. J Chromatogr 1153, 235–258.
Carpentier SC, Dens K, Van den Houwe I, Swennen R, and Panis B (2007) Lyophilization, a practical way to store and transport tissues prior to protein extraction for 2DE analysis? Proteomics 7, 64–69.
Cho JH, Hwang HY, Cho MH, Kwon YK, Jeon JS, Bhoo SH, and Hahn TR (2008) The effect of DTT in protein preparations for proteomic analysis: Removal of a highly abundant plant enzyme, ribulose bisphosphate carboxylase/ oxygenase. J Plant Biol 51, 297–301.
Delahunty C and Yates III JR (2005) Protein identification using 2D-LC-MS/MS. Methods 35, 248–255.
Dirnhuber P and Schütz F (1948) The isomeric transformation of urea into ammonium cyanate in aqueous solutions. Biochem J 42, 628–632.
Feiz L, Irshad M, Pont-Lezica RF, Canut H, and Jamet E (2006) Evaluation of cell wall preparations for proteomics: A new procedure for purifying cell walls from Arabidopsis hypocotyls. Plant Methods 2, 10–22.
Goodno CC, Swaisgood HE, and Catignani GL (1981) A fluorimetric assay for available lysine in proteins. Anal Biochem 115, 203–211.
Kim ST, Yu S, Kim SG, Kim HJ, Kang SY, Hwang DH, Jang YS, and Kang KY (2004) Proteome analysis of rice blast fungus (Magnaporthe grisea) proteome during appressorium formation. Proteomics 4, 3579–3587.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Lippincott J and Apostol I (1999) Carbamylation of cysteine: A potential artifact in peptide mapping of hemoglobins in the presence of urea. Anal Biochem 267, 57–64.
McCarthy J, Hopwood F, Oxley D, Laver M, Castagna A, Righetti PG, Williams K, and Herbert B (2003) Carbamylation of proteins in 2-D electrophoresis: myth or reality? J Proteome Res 2, 239–242.
Park SY, Milgroom MG, Han SS, Kang SC, and Lee YH (2003) Diversity of pathotypes and DNA fingerprint haplotypes in populations of Magnaporthe grisea in Korea over two decades. Phytopathology 93, 1378–1385.
Righetti PG (2006) Real and imaginary artefacts in proteome analysis via two-dimensional maps. J Chromatogr B 841, 14–22.
Sarnighausen E, Wurtz V, Heintz D, Van Dorsselaer A, and Reski R (2004) Mapping of the Physcomitrella patens proteome. Phytochemistry 65, 1589–1607.
Soulie S, Denoroy L, Caer JPL, Hamasaki N, Groves JD, and Maire ML (1998) Treatment with crystalline ultra-pure urea reduces the aggregation of integral membrane proteins without inhibiting N-terminal sequencing. J Biochem 124, 417–420.
Stark GR (1965) Reactions of cyanate with functional groups of proteins. III. Reactions with amino and carboxyl groups. Biochemistry 4, 1030–1036.
Stark GR, Stein WH, and Moore S (1960) Reactions of the cyanate present in aqueous urea with amino acids and proteins. J Biol Chem 235, 3177–3181.
Syrový I and Hodný Z (1991) Staining and quantification of proteins separated by polyacrylamide gel electrophoresis. J Chromatogr B Biomed Sci Appl 569, 175–196.
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Jeon, YT., Ruzicka, M.R., Cho, I.K. et al. Heating of freeze-dried protein samples with urea for SDS-PAGE in proteomics study. J. Korean Soc. Appl. Biol. Chem. 54, 19–23 (2011). https://doi.org/10.3839/jksabc.2011.002
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DOI: https://doi.org/10.3839/jksabc.2011.002