Lidstrom:Reducing Agents
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Why are reducing agents used?
Why are reducing agents a problem?
- Recombinant proteins with highly reactive thiol groups can form disulfide adducts with reducing agents commonly used in protein purification, such as beta-mercaptoethanol and dithiothreitol. These adducts can interfere with protein-protein or protein-ligand interactions.
What reducing agents are commonly used?
- DTT
- the most popular reducing agent for proteins (citation)
- b-ME (β-mercaptoethanol)
- foul odor, liquid
- widely used in prior to 1970 before being replacing by DTT (citation)
- TCEP (tris(2-carboxyl)phosphine)
Misc
- Side by Side comparison, DTT VS TCEP
- TCEP is not very stable in phosphate buffers, especially at neutral pH.
- Effective pH range is 1.5 to 8.5.