The primary reason for using DTT or DTE over 2-ME is that 2-ME is more volatile and therefore stinks more than either DTT or DTE. A secondary reason is that both DTT and DTE carry 2 sulfhydryl moieties, whereas 2-ME carries only one, therefore you use DTT and DTE at much lower concentrations, since disulfide bridge reduction requires 2 reactive sulfhydryls to break the -S-S- bond. The concentration difference between a monomolecular reaction and a dimolecular reaction is more like an order of 10 than 2 - i.e., if you use 1 micromolar DTT, use 10 micromolar 2-ME.

When oxidized, DTT forms a 6-member ring in which both hydroxyls can be equitorial, which is a lower energy state than when the hydroxyls are axial. With DTE, one of the hydroxyls will always be axial, and less free energy is released, which means the Keq is less than with DTT. The primary reason for using 2-ME is that it is cheaper than either DTT or DTE.

Hi. Both BME and DTT are reducing agents that inactivate disulfide bonds in ribonucleases. Therefore, they are equivalent. In my experience, DTT is easier to use because...it smells less. But, if you have a chemical fume hood and woork fast, you shouldn't have problems in the lab using beta mercapto ethanol.

Bye

Dear Aamir,

In general DTT is preferred over b-ME as it smells less and has double the reducing activity. Both are used in lysis buffer for 'protein extraction' but as you want to extract DNA there is no need to add either of them. Other components such as SDS etc are enough for the lysis and DNA extraction.

best ,

Sudhir

The primary reason for using DTT or DTE over 2-ME is that 2-ME is more volatile and therefore stinks more than either DTT or DTE. A secondary reason is that both DTT and DTE carry 2 sulfhydryl moieties, whereas 2-ME carries only one, therefore you use DTT and DTE at much lower concentrations, since disulfide bridge reduction requires 2 reactive sulfhydryls to break the -S-S- bond. The concentration difference between a monomolecular reaction and a dimolecular reaction is more like an order of 10 than 2 - i.e., if you use 1 micromolar DTT, use 10 micromolar 2-ME.

When oxidized, DTT forms a 6-member ring in which both hydroxyls can be equitorial, which is a lower energy state than when the hydroxyls are axial. With DTE, one of the hydroxyls will always be axial, and less free energy is released, which means the Keq is less than with DTT. The primary reason for using 2-ME is that it is cheaper than either DTT or DTE.

I can not assign more than one UpVote to Gary Gilmore answer but it is true and comprehensive answer. Price of reagents is does not matter in such biochemistry.

DTT can form adducts through S-S bond, B-Mercapto can not. B-Mercapto as stated above smells because of its volatility, but in my opinion is the safer to use when reducing proteins. For DNA, probably doesn't matter, shouldn't need it?

Dear Ryan, glad to find an real profi, request from lamer - can you give some references about "form adducts through S-S bond, B-Mercapto can not "?

And second question - have you experience in application of TCEP http://www.sigmaaldrich.com/catalog/product/sigma/75259 "..odorless, a more powerful reducing agent, an irreversible reducing agent , more hydrophilic, and more resistant to oxidation in air.." ?

I am sure that with TCEP we can solve all problems.

BME can crosslink two proteins if first it reacts with one protein to form a disulfide via oxidation, and then a second molecule comes in and react with the betamercaptoethanol-protein S-S bond to form a proteinS-Sprotein dimer.

MORTAL COMBAT announcement : Andrei vs Andrei!

First team is Andrei Nikonov - Ryan Workman, I take in players - Harry, Theo and Marcia.

Because the formation of a cyclic disulfide from DTT is much favorable because of the entropy factor and is much faster than the diffusion of a second molecule of BET, SS-bridging is practically excluded. But this process is known by BEM reductin for the two protein molecules, as well as the residual protein-SS-CH2CH2OH-rests as side-reactions - see Theo, Harry and Marcia answers. So the situation may reverse completely than told Ryan.

However, it is easy mistaken in science without having the facts, so I asked Ryan to give a citation. Perhaps he had in mind some metal complexes that are part of enzymes, or something else.

To our team pleasure, you scored an own goal. I looked at the link, and it became clearer why biochemists do not use TCEP. It is plainly how you made answer - copy-paste, experiment was not conducted did not read the article. It is surprising that such nonsense can be found at MIT, and replicated, settling in the minds of students - and then they will teach someone else.

Fortunately, MIT cited an source of instructions - this is GE Healthcare. They have good graphics for TSEP, but there do not all by hands and article did not read, or read but not understood. Exact phrase from article : "TCEP is NOT ONLY very stable in acidic solutions, BUT unlike dithiothreitol (DTT) which readily oxidizes above pH 7.5, it IS ALSO highly stable in basic solutions" - end of citation. http://www.sciencedirect.com/science/article/pii/S0003269784712905

WE ARE WINNERS! ;-)

More seriously, TCEP is 3-rd generation of SS-reductant, and I have ideas to 3++ generation - more faster, more safe and in times less expensive than TCEP - but I do not know this market, and can not imagine how to defend the idea from illegal copying because there is a very simple chemistry. I would be grateful if someone tell approach.

I used TCEP in PBS, and it worked fine. It is just unstable from what I have read, in phosphate buffer. I don't know the chemistry behind it. But I am at a loss as to why DTT or BME is used for DNA extraction? I don't work with DNA, but it would seem to me that TCEP would work, if the extraction method does not take long.

Both DTT and 2ME work fine for breaking disulfide bonds in hair keratin (not collagen) and thus breaking its structure, which facilitates hair DNA extraction.

Thanks for the answer as to why reducing agents are needed, Hieronim and Andrei.

thing is that the efficiency of DTT is more then BME. But problem with DTT is that its stability is less compared to BME.

TCEP is applied as phosphonium salt HP(+)(CH2CH2COOH)3 Cl(-) (sometimes as ester instead carboxyl) - this is very stable compound that is standard catalog item.

In addition to citation from my previous message for Andrei Nikonov, please read file in attachment. Only reason of instability of TCEP in PBS is slow autoxidation and it can be avoided by use of degassed solutions.

At moment of that publication, price for TCEP was 100x more that DTT. Now TCEP is slightly more costly than DTT (on mol basis):

http://www.sigmaaldrich.com/catalog/product/sigma/43815

http://www.sigmaaldrich.com/catalog/product/sigma/75259

This is autooxidation (check Hetz publication and "autooxidation" in Wiki) - because of presence of oxygen. Light and traces of heavy metals (substantially Cu, Mn, Fe) are initiators. In the case of heavy metals, this is variant of Fenton oxidation ( but with O2 instead of H2O2; Cu is 4x more productive than Fe). Complexation is not always possible (enzymes) and effective - if change of oxidation number (level) is possible.

Because autooxidation is free-radical process, it is less dependent from pH (but pH influence on oxidation potential of metals). In real life, in terms of practical utility, TCEP was investigated most in PBS condition.

Thanks to all of you wonderful researchers and scientist from around the globe for the help. And the answers. I really appreciate it, would love to hear from you guys and would keep in touch.

is anyone here familiar with DNA extraction from blood by beta-mercaptoethanol?