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TCEP – a reagent of choice in both Proteomics and nucleic acid chemistry

The sulfhydryl reductant tris (2-carboxyethyl) phosphine hydrochloride (TCEP) is a reagent of choice in protein biochemistry. TCEP is well-known to be superior to dithiothreitol (DTT)1. It’s higher thermal stability, better stability of the aqueous solution over a broader pH range, its suitability for long term storage of proteins, and its odorless characteristics, have made it a popular reagent in Proteomics.

Use of TCEP in nucleic acids is also exciting. It is found to stabilize RNA much better than DTT. For example, the half-life of full length RNA was extended by more than double2, 3. Its significantly higher thermo stability, combined with its ability to stabilize RNA against hydrolysis makes TCEP a reagent of choice in nucleic acid chemistry4. TCEP is used efficiently to generate covalently immobilized DNAs for in vitro transcription/ translation reactions5

TCEP.HCl is odorless so reactions can be carried out on the bench without the need for a fume hood. TCEP.HCl may be used as a substitute for DTT at a final concentration of 50 mM.

TCEP could be suitable for high through-put DNA sequencing application.

Polyorganix offers high quality TCEP [CAS# 51805-45-9] in gm to bulk quantities at most competitive price.
Specification: Assay 98% Min.

Contact us for a free samples,
pricing information, and bulk quantity inquiries.
We also offer Aq. Solution. Please contact Polyorganix, Inc.
information@polyorganix.com.
8159 Almeda Rd, Houston, TX 77054. Phone: 713-747-1887.

Advantage:

  • Molecular Biology Grade
  • Protease Free

Stability:

  • Odorless and non-volatile 6
  • Solid is stable in air for several months.
  • Dilute solutions in air show no appreciable oxidation up to at least 72 hours pH<7.66.

Reactivity:

  • Reduces organic disulfides to thiols rapidly and quantitatively in water7-8.
  • More complete reductions than 1,4-dithiothreitol ( DTT), Cleland's reagent.
  • Even very stable alkyl disulfides are completely reduced at room temperature and pH 5 in less than 5 min 9-11.
  • Dilute (1mM) TCEP solutions react rapidly at room temperature
  • The strength of the phosphorus-oxygen bond makes the reaction irreversible12.
  • Kinetics rather than thermodynamics controls the reduction.
  • Use ion making fluorescent protein tracers1.

Selectivity:

  • Selective for disulfides 13-15.
  • Does not react with other functional groups on proteins
  • Unreactive towards many common alkylating reagents so reductions have been carried out simultaneously with alkylations.

Potential use as a therapeutic agent

  • TCEP treatment reduces proteolytic activity of BoNT/B in humans.

TCEP, is a relatively non-toxic, non-sulfur containing disulfide bond reducing agent that lacks the undesirable properties of mercapto-containing reducing agents. Since disulfide bond coupling between toxin subunits is a general motif for many toxins, e.g., ricin, snake venom, and all BoNT serotypes, this suggests that TCEP is a promising means to protect against these toxins by preventing cell penetration 16.

References

1. Reaction of Tris(2-carboxyethyl)phosphine (TCEP) with Maleimide and a-Haloacyl Groups: Anomalous Elution of TCEP by Gel Filtration. Getz, Elise Burmeister, et.al, Analytical Biochemistry, 273, 73–80, 1999.

2. Tris(2-carboxyethyl)phosphine stabilization of RNA: comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry. Rhee, S.S., Burke, D.H., Analytical Biochemistry, 325, 137-43, 2004.

3. Turnover analysis of glutamate receptors identifies a rapidly degraded pool of the N-methyl-D-aspartate receptor subunit, NR1, in cultured cerebellar granule cells. Huh. K. H., Wenthold, R. J. Journal of Biological Chemistry, 274, 151-157, 1999.

4. A procedure for in situ alkylation of cystine residues on glass fiber prior to protein microsequence analysis. Andrews, P. C., Dixon, J.E., Analytical Biochemistry, 161, 524-528, 1987.

5. Use of immobilized PCR primers to generate covalently immobilized DNAs for in vitro transcription/translation reactions, oanne D. Andreadis and Linda A. Chrisey, Nucleic Acids Research, 28, 2, 5, 2000.

6. A procedure for quantitative determination of tris-(2-carboxyethyl)-phosphine, an odorless reducing agent more stable and effective than dithiothreitol. Han, J.C. and Han, G.Y. Analytical Biochemistry. 220, 5-10, 1994.

7. Reduction and fluorescent labeling of cyst(e)ine-containing proteins for subsequent structural analyses. Kirley, T.L., Analytical Biochemistry. 180, 231-236, 1989.

8. Selective reduction of disulfides by tris-(2-carboxyeth yl)-phosphine. Burns, J.A., Journal of Organic Chemistry, 56, 2648-2650, 1991.

9. Structure-reactivity relations for thiol-disulfide interchange. Houk, J., Whitesides, G.M., Journal of American Chemical Society, 109, 6825-6836, 1987.

10. Nucleophilic cleavage of the sulfur-sulfur bond by phosphorus nucleophiles. IV. Kinetic study of the reduction of alkyl disulfides with triphenylphosphine and water Overman, L.E., O'Conner, E.M., Journal of American Chemical Society, 98, 771-775, 1977.

11. Directional preferences of nonbonded atomic contacts with divalent sulfur. 1. Electrophiles and nucleophileFirst PageRosenfield, R.E., Parthasarthy, R., Dunitis, J.D., Journal of American Chemical Society, 99, 4860-4862, 1977.

12. The Cyanoethylation of Phosphine and Phenylphosphine. Rauhut, M., et. al., Journal of American Chemical Society, 81, 1103-1107, 1959.

13. Reduction and fluorescent labeling of cyst(e)ine-containing proteins for subsequent structural analyses. Kirkley, T.L., Analytical Biochemistrty, 180, 231-236, 1989.

14. Reductive cleavage of cystine disulfides with tributylphosphine. Ruegg, U.T. and Rudinger, J., Methods in Enzymology. 47, 111-126, 1977.

15. Tris-(2-carboxyethyl)-phosphine- A reducing agent with versatile applications including cleavage of disulfide bonds and quantitation of numerous oxidants. Han, J., et. al. Previews. 2, 16-21, 1999.

16. TCEP treatment reduces proteolytic activity of BoNT/B in human neuronal SHSY-5Y cells. Xuerong Shi, Gregory E. Garcia, Roger J. Neill, Richard K. Gordon, Journal of Cellular Biochemistry, 107, 5, 1021 – 1030, 2009.

 

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