Why does adding 10mM DTT to protein that contain 6 cys become yellow?
Dear Wafa,
The yellow color is a result of the reaction of DDT with the protein:
DTT is frequently used to reduce the disulfide bonds of proteins and peptides. It prevents intramolecular and intermolecular disulfide bonds from forming between cysteine residues of proteins. However, even DTT cannot reduce buried (solvent-inaccessible) disulfide bonds, so reduction of disulfide bonds is sometimes carried out under denaturing conditions (e.g., at high temperatures, or in the presence of a strong denaturant such as 6 M guanidinium hydrochloride, 8 M urea, or 1% Sodium dodecylsulfate). Conversely, the solvent exposure of different disulfide bonds can be assayed by their rate of reduction in the presence of DTT. DTT is as a reducing or “deprotecting” agent. DTT protects notably enzyme activity loss by the oxidation of sulfhydryl groups. So DTT is widely used in biochemistry works to reduce dissulfide bridges, protect biomolecules, in sample preparation, and to denature proteins before electrophoresis analysis (SDS-PAGE). The DTT removal is performed by standard desalting procedures (dialysis, gel filtration).
http://agscientific.com/blog/index.php/2013/01/11/dithiothreitoldtt-application-you-must-know/
Hoping this will be helpful,
Rafik
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