Dithiothreitol dtt is a redox reagent also known as clelands reagent. The neutral ph of this reagent provides sharp bands and avoids. Tcep was found to cleave protein backbones under mild conditions by. Tcep is a potent versatile odorless thiolfree reducing agent with many applications in protein chemistry and proteomic research centered in the quantitative ease of reducing disulfide bonds. Oxetane grafts installed sitea selectively on native. For longterm storage of proteins, tcep is significantly more stable than dtt without metal chelates such as egta in the buffer, whereas dtt is. The versatile compound is readily soluble and very stable in aqueous solutions. Compared to dtt, tcep is more stable, more effective and able to reduce disulfide bonds at lower phs. In a recent analysis of the factors contributing to the stability of rnase t1, the stabilizing and destabilizing. Reducing all five disulfide bonds inactivates the aspergillus niger phytase due to conformational change andor unfolding. Oxetane grafting of the genetically detoxified diphtheria toxin crm 197 improves significantly the immunogenicity of this protein in mice, which illustrates the general utility of this strategy to modulate the stability and biological activity of therapeutic proteins containing disulfides in their structures. The resin allows for the efficient reduction of peptide and. Tcep tris2carboxyethylphosphine is a strong reducing agent.
Dtt becomes less a less potent reducing agent with decreasing ph levels. This allows you to prepare tcep working concentrations and 10x stock solutions in the buffer of your choice before use. General description tris2carboxyethylphosp hine hydrochloride tcep. How can i purify reduced protein to get rid of the reducing.
Thermo scientific bondbreaker tcep solution, neutral ph is a stable, 0. The resin allows for the efficient reduction of peptide and protein disulfide bonds. Tcep alters in a lower extend 1020% iodoacetamides reactions with sufhydryls when at low concentrations, but stronger interference is observed at higher concentrations. How can i purify reduced protein to get rid of the. The unnatural disulfide bond was engineered at the surface of the protein, whereas the natural disulfide bond is buried in the protein interior. Thus tcep is a useful reductant over a much wider ph range 1. This product is an effective and convenient replacement for bmercaptoethanol or dtt in sdspage sample buffers. Gbiosciences tcep reducing resin consists of tris2carboxyethyl phosphine hydrocholride covalently coupled to 4% crosslinked beaded agarose. L flowthrough 1 vol pp rt, 10 min 5 min, 11,000 x g wash protein pellet 500. A tris 2carboxyethyl phosphine tcep related cleavage on.
Tcephcl, tris2carboxyethylphosphine hydrochloride, 1g. A comparison between the sulfhydryl reductants tris2. It is a toxic and carcinogenic compound that is frequently detected in wastewater effluents and water bodies, including those used for drinking water, around the world. The project includes theoretical chapters describing proteins, amino acids, protein stability, circular dichroism spectroscopy, and steadystate.
Tris2carboxyethylphosphine hydrochloride powder sigma. Jan 14, 2017 1 reconstitute dried protein sample in 40 ul of 8. Tcep hydrochloride can be a viable alternative to dtt as a reducing agent in crystallization set ups. May 24, 2016 the unnatural disulfide bond was engineered at the surface of the protein, whereas the natural disulfide bond is buried in the protein interior.
Protein reduction, alkylation, digestion last updated 1042011 page 3 reduction alkylation dtt, iaa and rapigest or pps, trypsin digestion. The rational modification of the structure of peptides and proteins offers a wide range of opportunities for the modulation of their biological activity. It reduces disulfides at room temperature in tcep prior to the use of sulfhydrylreactive labels or crosslinkers. Tcep effectively reduces disulfide bonds over a broad ph range. The chemical reagents mentioned above are the most commonly used to extact recombinant proteins but it should be mentioned that in experminental protein folding to determined mechanisms and pathways, often temperature is used to unfold proteins. Thermo scientific bondbreaker tcep solution, neutral ph. Material may be irritating to the mucous membranes and upper respiratory tract. Protein reduction can be achieved within 30min with 23m for aquous solutions of 210mg protein ml levison. In addition, tcep has been advertised as being unreactive with thiolreactive compounds, thereby elimi. Tcep is stable in aqueous, acidic, and basic solutions. It reduces disulfides at room temperature in stability the product, as supplied, is stable for at least two years e product is opened, any unused material should be flushed with argon and for 1 week at room temperature.
Works well for complex mixtures to be analyzed by mass spec. This polymerization mixture will be a mix of about 15% biotinylated and 15% dyelabeled protein if being used for tirf, or 100% biotinylated hydrolysis dead mutant if for em. Bondbreaker tcep solution is a potent, odorless, thiolfree reducing agent with broad application to protein and other research involving reduction of disulfide bonds. The protein ingel tryptic digestion kit is designed for colloidal coomassie or fluorescent dyestained acrylamide gel slices.
For protein bands stained with mscompatible silver stains or reversible zinc. Tcep tris2carboxyethylphosphine is a reducing agent frequently used in biochemistry and molecular biology applications. The effect of the disulfide bridges on the structural stability of the. Tcep hcl is odorless, airstable crystalline solid, soluble in water at a 1 m concentrations.
While tcep can be used in phosphate buffers, it has the tendency to be unstable around neutral. A comparison between the sulfhydryl reductants tris 2. L psbtcep 3 min, 95981 c 1 min, 11,000 x g dna and rna. It is often prepared and used as a hydrochloride salt tcep hcl with a molecular weight of 286. Currently, the most popular reducing agent for proteins is dithiothreitol dtt. Dtt is a small molecule and is an epimeric compound of dithioerythritol dte these reducing reagent products are readily supplied by ag scientific, inc. Procedure sdspage sample preparation with tcep dilute the 0. It is a toxic and carcinogenic compound that is frequently detected in wastewater effluents and water bodies. A crystalline solid laboratory procedures for long term storage, we suggest that tcep hydrochloride be stored as supplied at 20c. The role of thiols and disulfides in protein chemical and. In comparing the effects of phosphate buffer ph on tcep stability, han and han found the greatest tcep oxidation between ph 7. The thermo scientific tcep is a potent, versatile, odorless, thiolfree reducing agent with broad application to protein and other research involving reduction of disulfide bonds figure 1. The use of reducing agents the presence of a reducing agent in experimental solutions is often necessary for some proteins to maintain activity and stability andor prevent it from forming aggregates. Pdf a comparison between the sulfhydryl reductants tris2.
Storage stability the product, as supplied, is stable for at least two years e product is opened, any unused material should be flushed with argon and for 1 week at room temperature. Protein purification protein in the column flowthrough 12 precipitate protein 10700. This allows you to prepare tcep working concentrations and 10x stock solutions in. It is often prepared and used as a hydrochloride salt tcephcl with a molecular. Thermo scientific pierce tcep hcl is a potent, versatile, odorless, thiolfree reducing agent with broad application to protein and other research involving reduction of disulfide bonds. The unique compound is easily soluble and very stable in many aqueous solutions. Enhancing protein stability with extended disulfide bonds. Polymerize protein with excess nucleotide in the presence of equimolar or slightly excess crosslinking reagent for 30 min e. Tcep hcl is an alternative reducing reagent which is more stable and works even at low ph. T he rational modification of the structure of peptides and proteins offers awide range of opportunities for the modulation of their biological activity. Furthermore, this project includes a state of the art chapter, which concerns with fatal protein misfoldings. It is soluble in water and available as a stabilized solution at neutral ph and immobilized onto an agarose. Tcep hydrochloride is supplied as a crystalline solid.
It has been demonstrated that the bulky reducing reagent tris2carboxyethylphosphine tcep preferentially reduces exposed versus buried disulfide bonds. It is used to break down protein disulfide bonds and stabilize enzymes and other proteins. Efficient for most applications, 5 to 50mm tcep provides sufficient molar excess to effectively reduce peptide or protein disulfide bonds within a few minutes at room temperature. Reducing all five disulfide bonds inactivates the aspergillus niger phytase due. I know tcep is a better reducing agent than dtt because tcep can react without being in competition with the maleimide. For protein bands stained with mscompatible silver stains or reversible zinc staining, alternative destaining procedures will be required 3, 4. Intramolecular disulfide bonds are known to contribute to protein thermal stability. L ethanol 50 % 1 min, 11,000 x g 14 dry protein pellet rt, 510 min 15 prepare protein sample 20100. A novel methodology for assignment of disulfide bond. Tcep tris 2carboxyethylphosphine is a reducing agent frequently used in biochemistry and molecular biology applications.
It reduces disulfides at room temperature in tcep is a biorecalcitrant flame retardant and plasticizer. This project will introduce the topic of protein folding. Tcep hydrochloride is an odorless, thiolfree reagent useful for the selective reduction of disulfides. Use of protein folding reagents pubmed central pmc. In a recent analysis of the factors contributing to the stability of rnase t1, the stabilizing and destabilizing interactions were estimated at 271 and 286 kcalmol, respectively pace et al. Understanding the impact of methionine oxidation on the. Tcep protein expression purification reagents products. Also, ph is another important parameter in protein stability and solubility. Stability in solution tcep is stable in aqueous, acidic, and basic solutions.
Storagestability the product, as supplied, is stable for at least two years e product is opened, any unused material should be flushed with argon and for 1 week at room temperature. A stock solution may be made by dissolving the tcep. Enhancing protein stability with extended disulfide bonds pnas. This is a major reason for the difficulty of quantitative computational calculation of protein stability. The unique compound is easily soluble and very stable in. We compare properties of dtt and tcep im portant in protein biochemistry, using the motor en. Stable in air the inherent stability of the tcep moiety eliminates the need for any special precautions to avoid oxidation when handling, using or storing tcep. In fact tcep is stable in aqueous, acidic and basic solutions. Tcep can be dissolved in many types of aqueous buffers, and at a wide range of ph levels and still maintain stability.