Lidstrom:Reducing Agents

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)
  • more expensive
  • lacks odor (citation)
  • stronger reducing capacity and decreased probability of oxidation in air (citation)

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.