Article Contents
Sulfhydril Groups and Disulfide Bridges in Proteins
How to Prepare 0.5 M TCEP Stock Solution
What is TCEP?
Tris (2-carboxyethyl) phosphine (TCEP) is a reducing reagent used in molecular biology and protein biochemistry research.
TCEP Chemical Structure (PubChem ID: 119411)
Researchers often add TCEP to denature proteins during preparation of protein samples for gel electrophoresis. TCEP has also been useful for long-term storage of protein and preparation of samples in many other applications, such as:
- Protein conjugation or labeling
- Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF)
- Protein purification including chromatography
- During DNA and RNA isolation
Sulfhydril Groups and Disulfide Bridges in Proteins
Peptides and proteins consist of amino acids linking together in peptide bonds.
Sulfhydryl groups or thiols (R-SH) are present in proteins containing amino acid cysteine residues. When two sulfhydryl groups are close to each other, they can form a disulfide bridge (R-S-S-R’) by an oxidation reaction.
The repeated groups of cysteine residues and disulfide bridges are commonly present in extracellular domains of membrane-bound receptors. Many extracellular or secreted proteins and peptides also have disulfide bridges, including some hormones, enzymes, plasma proteins, inhibitors, and venom proteins. This disulfide bridge structure is important for the biological function of protein and stabilization of the secondary and tertiary structure of a protein.
Denaturation of a Protein. Addition of a reducing agent cleaves disulfide bridges and denatures a protein.
To study the protein structure and function, researchers often analyze each disulfide bridge as one enzymatic fragment and study how one bridge connects to the others. When the cysteine residues are closely grouped, it is hard to separate the peptide chain between those disulfide bridges.
Adding a reducing agent to break disulfide bonds is one way to solve this problem. A reducing agent is a compound that donates an electron (or electrons) to another compound. Some examples of the reducing agents are TCEP, DTT, 2-mercaptoethanol, and 2-mercaptothylamine.
TCEP and Reduction Reactions
TCEP is an effective reagent for the cleavage of disulfide bridges. TCEP is stable in aqueous solutions, highly reactive, and selective towards disulfide structure.
There are two main steps in the breaking of disulfide bridges and forming a free sulfhydryl using TCEP:
- cleavage of the S-S bond
- oxidation of the phosphine and release of sulfhydryl
TCEP Reaction. 1. Cleavage of S-S bond and formation of a compound containing a disulfide bridge structure. 2. Release of sulfhydryl molecules and formation of oxidized phosphine.
TCEP or DTT?
- Compared to DTT and other reducing agents, TCEP has a more neutral odor and TCEP is more resistant to oxidation by air.
- TCEP is a useful reductant with a wide pH range (1.5-8.5) and it is more stable than DTT at pH above 7.5 (biological pH).
- TCEP reaction is irreversible, whereas DTT reaction is reversible.
- Compared to DTT, TCEP is preferred for labeling cysteine residues with maleimides. DTT contains thiols and shows reactivity with maleimides, which reduces the labeling efficiency.
- You can use either TCEP or DTT to label with iodoacetamide. TCEP and DTT only lower labeling efficiency of iodoacetamide by a very small amount. But the ratio of dye over iodoacetamide, pH, and temperature may affect the efficiency of iodoacetamide labeling.
- TCEP is more stable than DTT in the presence of metal ions (such as Fe3+ and Ni2+). You can use TCEP instead of DTT to perform metal-ion affinity chromatography.
- In the presence of EGTA, DTT’s stability increases, but TCEP’s stability decreases. The TCEP instability is due to metal chelators (such as EGTA) catalyzing TCEP oxidation.
- You can use either TCEP or DTT to label with iodoacetamide. TCEP and DTT only lower labeling efficiency of iodoacetamide by a very small amount. But the ratio of dye over iodoacetamide, pH, and temperature may affect the efficiency of iodoacetamide labeling.
Note: TCEP does not contain thiols so there is no need to remove it from the labeling reaction. But TCEP may react with maleimides under certain conditions, such as acidic conditions, at 20°C, and too much TCEP for labeling of proteomic samples.
If you decide to remove excess TCEP before the addition of maleimides, you can use:
- dialysis
- TCEP-immobilized resin
- column chromatography
-4-azidobenzoic acid
Buffers to Dissolve TCEP
- You can use many types of common buffers during preparation of protein samples to dissolve TCEP at a wide range of pH levels.
- TCEP tends to be unstable around neutral pH in phosphate buffers. If you need to use phosphate buffers with TCEP, prepare it immediately before use
For examples, you can prepare TCEP in:
- Tris-HCl buffer for protein labeling or protein purification
- Borate buffer for capillary electrophoresis-laser induced fluorescence
- Hepes buffer for chromatography
How to Prepare 0.5 M TCEP Stock Solution
1.Weigh 5.73 g of TCEP (TCEP-HCL, GoldBio Catalog # TCEP)
2.Add 35 ml of cold molecular biology grade water to the vial, and dissolve the TCEP. This resulting solution is very acidic, with an approximate pH of 2.5.
3.Bring the solution to pH 7.0 with 10 N NaOH or 10 N KOH.
4.Bring the resulting solution to 40 ml with molecular biology grade water.
5.Aliquot into 1 ml into freezer tubes and store at -20°C.
Note: This protocol allows you to prepare TCEP working concentrations and 10X stock solutions in the buffer of your choice before use. You can use TCEP as a substitute for DTT at a final concentration of 50mM.
Note: TCEP cannot be used for isoelectric focusing due to its charge in solution.
Note: Cover the tubes with aluminum foil, because TCEP is light sensitive.
Note: Stock solutions are stable for 3 months at -20°C.
Related Product
TCEP-HCL, GoldBio Catalog # TCEP
Related Articles
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