Product FAQ

Why is Beta-mercaptoethanol (BME) no longer included with the One Shot Chemically Competent E. coli kits? What was the purpose of the BME during E. coli transformation?

Answer

Beta-mercaptoethanol (BME) degrades carbohydrates on the cell surface, which theoretically allows DNA to get closer to the membrane prior to heat shock. In the past, this was thought to improve the efficiency of transforming E. coli strains, and the addition of Beta-mercaptoethanol was a standard practice for all chemical transformations. However, galU and galK minus strains, such as TOP10, INV?F', DH5?, DH10B and TOP10F', have fewer carbohydrates on the cell surface. After repeated testing of all of our strains, we determined that adding BME had no beneficial effect on transformation efficiency, and we chose to remove BME from the chemically competent One Shot kits.

Answer Id: E3359

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Product FAQ

Can beta-mercaptoethanol (BME) be used rather than DTT as the reducing agent in the NuPAGE LDS Sample Buffer?

Answer

Either BME or DTT can be used in the NuPAGE LDS Sample Buffer.

Make sure that a fresh solution of BME is used. FINAL concentration:

DTT 50-100 mM

BME 2-5%

Answer Id: E3836

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Product FAQ

Can other reducing agents other than DTT or BME be used to reduce proteins prior to electrophoresis? For example, what about TCEP (Tris Carboxy Ethyl Phosphene)?

Answer

TCEP, Tris Carboxy Ethyl Phosphene is an alternative sulfhydryl reducing agent for protein samples. It is an extremely potent and effective reducing agent for particularly ‘difficult’ proteins. It is compatible with the Tris-Glycine gels and NuPAGE gels. It should be added to the sample buffer for these systems. 20 mM final (maximum) concentration is sufficient for samples. You may add alkylating agents, e.g. Iodine (50 mM Iodoacetic acid), to prevent re-forming of S-S bonds but it is not necessary. Do not heat because this will hydrolyze much of your sample. Instead let the sample sit for several minutes at RT and then load.

Answer Id: E3577

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Product FAQ

A protein sample with many disulfide bonds, reduced with BME or DTT, is exhibiting smeary artifacts on a Tricine Gel. Are the samples insufficiently reduced?

Answer

One potential explanation is that the protein sample is getting re-oxidized before the run is complete. Reduced samples tend to oxidize more in the Tricine system. Adding more reducing agent will not solve the problem. One option is to alkylate the sample by reducing with 20 mM DTT at 70 degrees C for 30 minutes, followed by 50 mM iodoacetic acid. Another method which inhibits oxidation is the addition of thioglycolic acid to the running buffer. The reference to this is described by Hunkapiller et al., Methods in Enzymology, (91), 399, 1983. Caution should be taken when using this method since this compound is both toxic and expensive. In addition, the TGA must be fresh as it tends to get self-oxidized over time and will promote sample re oxidation.

Answer Id: E10779

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Citations & References

Micellar Enhanced Fluorimetric Determination of 1-N,N-Dimethylaminonaphthalene-5-sulfonyl Chloride and o-Phthalaldehyde-2-Mercaptoethanol Derivatives of Amino Acids.

  • Authors: Singh HN, Hinze WL
  • Journal: Analyst (1982) 107:1073-1073
Catalog #
  • D21(Discontinued)
  • P2331MP(Discontinued)

Product FAQ

Can I reduce proteins with dithiothreitol (DTT) or beta-mercaptoethanol before running on an IEF gel?

Answer

Yes, you can reduce the protein with DTT or beta-mercaptoethanol prior to running it on an IEF gel, keeping in mind that if the reducing unfolds or opens masked charges, you may see a change in pI.

Answer Id: E10643

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Citations & References

Derivatization of cysteine and cystine for fluorescence amino acid analysis with the o-Phthaldialdehyde/ 2-mercaptoethanol reagent

  • Authors: Lee, K., et al.
  • Journal: J Biol Chem July (1979) 25:6248-6251
Catalog #

Product FAQ

Can I use beta-mercaptoethanol instead of the NuPAGE Sample Reducing agent?

Answer

Although we recommend using the NuPAGE Sample Reducing agent for stability reasons, fresh, neat beta-mercaptoethanol can be substituted for the NuPAGE Sample Reducing Agent, with equivalent results. A final concentration of 2-5% beta-mercaptoethanol is usually sufficient to reduce the sample.

Answer Id: E10554

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Product FAQ

I've purchased Gibco 2-Mercaptoethanol (Cat. No. 21985023). Should I add it to my cell culture medium right before every use or can I add it to the bulk medium in advance?

Answer

The 2-Mercaptoethanol must be added to the medium immediately before use because activity rapidly declines in diluted form. You can supplement enough medium needed for that day.

Answer Id: E17388

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Product FAQ

What effect does DTT or beta-mercaptoethanol have on the ProBond resin?

Answer

DTT or beta-mercaptoethanol treatment causes the resin to be stripped of nickel ions. The reducing agents ‘attack’ the iminodiacetic acid (IDA) groups on the resin that chelate the nickel ions.

Answer Id: E3669

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Citations & References

The Oxidative Addition Reaction between Compounds of Resorufin (7-Hydroxy-3H-phenoxazin-3-one) and 2-Mercaptoethanol

  • Authors: Kitson TM
  • Journal: Bioorg Chem (1998) 26:63-73
Catalog #
  • R352(Discontinued)
  • R6564(Discontinued)

Citations & References

Inactivation of (Na+,K+)-ATPase by beta-mercaptoethanol. Differential sensitivity to reduction of the three beta subunit disulfide bonds.

  • Authors: Kirley TL
  • Journal: J Biol Chem (1990) 265:4227-4232
  • PubMed ID: 2155215
Catalog #
  • F6053(Discontinued)

Citations & References

Effects of 2-mercaptoethanol on survival and differentiation of fetal mouse brain neurons cultured in vitro.

  • Authors: Ishii K, Katayama M, Hori K, Yodoi J, Nakanishi T
  • Journal: Neurosci Lett 1993 (163):159-162
  • PubMed ID: 8309623