Urea, 500g (C08-0503-731)

Urea is a prinicipal protein metabolite end product of nitrogen metabolism in most mammals, formed by the enzymatic reactions of the Kreb's cycle and the major product for the removal of free ammonia (NH4+) in vivo. Urea is a mild agent usually used in the solubilization and denaturation of proteins. It is also useful for renaturing proteins from samples already denatured with 6 M guanidine hydrochloride such as inclusion bodies; and in the extraction of the mitochondrial complex. It is commonly used to solubilize and denature proteins for denaturing isoelectric focusing and two-dimensional electrophoresis and in acetic acid-urea PAGE gels. Urea in solution is in equilibrium with ammonium cyanate. The form that reacts with protein amino groups is isocyanic acid. Urea in the presence of heat and protein leads to carbamylation of the proteins. Carbamylation by isocyanic acid interferes with protein characterization because isocyanic acid reacts with the amino terminus of proteins, preventing N-terminal sequencing. Isocyanic acid also reacts with side chains of lysine and arginine residues resulting in a protein that is unsuitable for many enzymatic digests. In addition, carbamylation often leads to confusing results from peptides having unexpected retention times and masses. Urea is used in cell or tissue culture media to increase the osmolality. Urea has also been used as fertilizer because of the easy availability of nitrogen; in animal feeds; it is reacted with aldehydes to make resins and plastics; condensed with malonic ester to form barbituric acid; used in the paper industry to soften cellulose; used as a diuretic; enhances the action of sulfonamides; an antiseptic. Used for the denaturation of proteins and as a mild solubilization agent for insoluble or denatured proteins. Useful for renaturing proteins from samples already denatured with 6 M guanidine chloride such as inclusion bodies. May be used with guanidine hydrochloride and dithiothreitrol (DTT) in the refolding of denatured proteins into their native or active form. Urea is typically used at a concentration of 8 M for protein denaturation or solubilization. A final concentration of 5 M urea is commonly used in molecular biology for sequencing gels. To prevent carbamylation, do not heat urea containing buffers above 37°C.