I'm not sure if this has been brought up already but TCEP has the advantage over DTT and B-ME that it does not reduce metals as easily as the other two. A significant advantage in IMAC if strong reducing conditions are needed. Also, TCEP has a bulky nature and thus it doesn't reduce intramolecular disulfides as easily as the other two, which is very nice if the protein contains disulfides that are essential for stability/correct folding.

I usually use mercaptans (DTT mainly) during purification due to their low cost but I store my proteins in TCEP because of its stability (unless I'm using phosphate buffer). Also, TCEP effectively keeps reducing conditions even at µM concentrations. 1 mM TCEP is quite high already...

Hi Tomas,

for IMAC we use 1 mM beta-ME, for SEC and/or crystallzation of proteins 1 mM DTT or 0.5-1 mM TCEP.

Cheers,

Magdalena

2-10mM BME for purification and most other things, 0.5-5mM DTT for storage buffer or when the actual concentration needs to be well known and fixed. 0.5-5mM TCEP or 2-50(yes, fifty)mM DTT for crystallization. Also good for crystallization: after mixing the drop add excess BME (~100mM) into the well solution and seal. It will diffuse into your sample.

Hi Tomas,

I find BME and DTT similarly good, but during electrophoresis I generally prefer BME (2-3% in the loading buffer).

I sometimes use also TCEP as it has the advantage of acting in a broader range of pH and of being odorless!

Cheers

Matteo

Hi!

TCEP and DTT are much more stable than BME, but also more expensive. So, for purification I use BME and than change to DTT (30 mM) or TCEP (2mM) for storage.

BME for initial stages of purification. cheap and effective but smelly.

DTT for later stages and storage as it is quite efficient but unstable as prone to oxidation. Use TCEP as an alternative. More stable than DTT but can be expensive. Caution! I advise you buy the pH'ed version or adjust after addition as will lower pH of your sample and avoid if using phosphate buffers.

If you can put up with the smell there is no reason that BME shouldn't be used.

So, you now know that it is a matter of preference (mostly) and please use what is readily available at the moment!

As to the concentration, it just need to be large and anything around 1 mM is large enough (please also consider the dissolved oxygen also).

@Andrew Mcewan - I've read contrary that bME is more prone to oxydation

@tomas sorry if i caused any confusion but I didn't say it wasn't and I'll stand by my comment and reitterate my feelings that Bme is a good all rounder.

Incidentally I was telling someone in the lab about this thread and couldn't think of any abetter lternatives to the bme, tcep and dtt. Would be good to know if anyone finds an alternative especially if they have used it.

According to Wikipedia: 2-mercaptoethanol is more stable than DTT (2-ME: t1/2>100h at pH6.5, t1/2=4h at pH8.5; DTT: t1/2=40h at pH6.5, t1/2=1.5h at pH8.5; but BME has a higher volatility, so I keep it in the 5oC frig. That is why I have always used BME for protein purification where the solutions are at 5oC for long periods of time (i.e., O/N dialysis).

Dtt

I'm not sure if this has been brought up already but TCEP has the advantage over DTT and B-ME that it does not reduce metals as easily as the other two. A significant advantage in IMAC if strong reducing conditions are needed. Also, TCEP has a bulky nature and thus it doesn't reduce intramolecular disulfides as easily as the other two, which is very nice if the protein contains disulfides that are essential for stability/correct folding.

I usually use mercaptans (DTT mainly) during purification due to their low cost but I store my proteins in TCEP because of its stability (unless I'm using phosphate buffer). Also, TCEP effectively keeps reducing conditions even at µM concentrations. 1 mM TCEP is quite high already...

The only reason I have not tried TCEP is the cost:

4 gr of TCEP (Pierce 10 gr/$356, $35/gr) can make 2.9L of a 5 mM solution.

1 mL of BME (Sigma, 500 mL/$70, 14 cents/mL) can make 2.9L of a 5 mM solution. When your are dialyzing a protein solution against 1L of buffer 3X (3L total) you use a lot of reducing agent (1 mL BME for 14 cents, versus 4 gr of TCEP for $142, that is a 1000-fold increase in price for TCEP versus BME).

DTT is also a lot more expensive than BME.

Though as Arne pointed out if you use TCEP just for protein stocks then you would not need that much and that would keep the cost down.

DTT (proteins), rarely mercaptoethanol (for small molecules)

I found this today -includes some alternatives.

https://webshop.fishersci.com/webfiles/uk/web-docs/UKLS_0698.PDF

Andrew, the document also lists the Fisher protein stabilizing buffer which is interesting, though I would still use 50% glycerol for -20oC storage.

I would recommend purchasing TCEP from the manufacturer Soltec - product page here: http://www.soltecventures.com/TCEPHCl-p-1632

They charge significantly less - $95 for 10 g.

Dear All

The main advantage of TCEP is that its absorb less in UV than the others. See:

http://www.uslims.uthscsa.edu/reductants.php

Therefore it is a good idea to use it you do any UV detection of your protein in your buffer. I will recommend it in the final buffer as it will disturb much less in analytical measurements.

Bertrand, this might be a follow-up question to the usage of TCEP in optical measurements.

Do you know if TCEP can be used when doing far-UV CD (240nm-180nm)? If so which concentrations and pathlength on kyvetts?

I have used only 2-Mercatpoethanol. But I have purified only bacterial proteins (Around 10). 2-ME works perfectly fine.

I agree with Arne Raasakka about the advantages of trying and using TCEP. In working with proteins such as nitric oxide synthase to make large amounts of protein for crystal structures, TCEP was often key for our success in obtaining high quality active protein and well-diffracting crystals.

I used DTT and Beta mercaptothanol but smetimes I also used Na bisulfite with certain protein in rare occasions The concentrations vary within mM

During protein purification I use DTT most frequently. However, DTT is not very stable and needs to be made fresh each time you use it. Also, at higher concentrations DTT is not compatible with some [protein] quantification techniques such as the BCA assay so you need to take that into consideration for your experiments.

TCEP HCl, DTT and BME have been shown to reduce protein aggregation that may inhibit crystallization.Reducing agents can interact with metals within your sample resulting in poor efficiency of your reducing agent. If possible it is suggested to use a metal chelating agent such as EDTA to remove all undesired metals. Unfortunately, this may not be possible since many enzymes require metal in order to properly function. BME is especially sensitive to copper, cobalt and many phosphate buffers. DTT is sensitive to nickel therefore caution should be used when if used in combination with affinity chromatography.

DTT has a shorter half-life than BME but has a low volatility. Both have a big dependence of pH. At pH 6.5 (20ºC) BME has a half-life greater than 100 hours and DTT 40 hours. At pH 8.5 (20ºC) half-lives reduce to 4 h and 1.4 h respectively (Stevens et al., 1983).

https://www.researchgate.net/publication/309041540_Important_Factors_Influencing_Protein_Crystallization

Article Important Factors Influencing Protein Crystallization

This link will really help you, 

http://instanano.com/characterization/theoretical/reducing-agent-calculator-nanoparticles-synthesis/

It depends on the structure of each proteins and effect of reducing agents on further downstream processing procedures. I always use DTT. It works fine for me.

I have been seeing BME adducts on cysteines in mass spec (unwanted artificial modification). I saw this being mentioned in Wikipedia too. So for such applications probably use DTT or maybe TCEP is better.

do we have to remove DTT before crystallization trials.

how much concentration of DTT is tolerable for crystallization procedures?

2-ME is good reducing agent in SDS-PAGE