Lidstrom:Reducing Agents: Difference between revisions
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== Why are reducing agents used? == | == Why are reducing agents used? == | ||
* Two cystines can either be paired in disulfide bonds (−S−S−) or can be reduced as two sulfhydryl groups (−SH). | |||
** If a cystine is catalytic, it needs to be in the reduced form. | |||
* Disulfide bonds in proteins are subject to cleavage by mild reducing agents, by a few oxidizing agents, or by nucleophilic displacement ([http://www.mechamlab.wustl.edu/Lab%20Web%20Page.data/Library/MethodsPDFs/Protein_Modification/Disulfide.pdf citation]) | |||
== 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. ([http://www.abrf.org/jbt/1999/March99/mar99beggspeicher.html citation]) | |||
== 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? == | == What reducing agents are commonly used? == | ||
* DTT | * DTT | ||
** the most popular reducing agent for proteins ([http://csbi.mit.edu/instrumentation/Reducing%20Agents.pdf citation]) | |||
* b-ME (β-mercaptoethanol) | * b-ME (β-mercaptoethanol) | ||
* TCEP | ** foul odor, liquid | ||
** more expensive | ** widely used in prior to 1970 before being replacing by DTT ([http://csbi.mit.edu/instrumentation/Reducing%20Agents.pdf citation]) | ||
* TCEP (tris(2-carboxyl)phosphine) | |||
** more expensive | |||
** lacks odor ([http://csbi.mit.edu/instrumentation/Reducing%20Agents.pdf citation]) | |||
** stronger reducing capacity and decreased probability of oxidation in air ([http://csbi.mit.edu/instrumentation/Reducing%20Agents.pdf citation]) | |||
== Misc == | == Misc == | ||
Latest revision as of 15:36, 24 April 2014
Back to Protocols
Why are reducing agents used?
- Two cystines can either be paired in disulfide bonds (−S−S−) or can be reduced as two sulfhydryl groups (−SH).
- If a cystine is catalytic, it needs to be in the reduced form.
- Disulfide bonds in proteins are subject to cleavage by mild reducing agents, by a few oxidizing agents, or by nucleophilic displacement (citation)
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. (citation)
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.
