Yes, DTT is heat sensitive and hygroscopic. I dont have handy numbers for stability of DTT is tris, but you can also use TCEP for dialysis (TCEP is more stable). Another possibility is to use 5 fold more DTT, and do the dialysis at 4C, and then change your buffer with required DTT conc in morning. If you want to measure then oxidized DTT forms peak at 310 nm, and your protein has negligible absorbance at this wavelength

TCEP might be a better option for you, if you are worried about stability of the reducing agency.

Dear Mahershawaran

first of all, can i ask you why you would like to work in presence of DTT? You proteins contain free cisteines that are important for his activity and not involved in the formation of any S-S bonds?

I the past i worked a lot with 2 kind of proteins,

1) proteins those contains S-S bonds (e.g Cu,Zn Sod1)

2) proteins that use cisteines to bind cofactors or for its catalitic activity (e.g Sco1, Trx, Sortases)

while for the second, the use of DTT is often necessary to activate the protein, for the first it is distruptive and if in our expression we are not able to produce the protein with S-S bonds generally is it preferable change the expression condiction, eg expression host, strain, leader sequence to produce protein in the correct state and not perform the purification in presence of reducing agents.

Reducing agenst seems to solve your problem. because protect protein from aggregation and precipitation but they also destoid protein structure and activity.

on the other hand if your protein belong to the second group you have to work with protein in reducing condiction, but it is very difficult because each reducing agent, if you are working in presence of air, will be re-oxidated.

One possibility as Mahesh suggest is use an excess of DTT at 4°C or TCEP (which r is little bit more stable) but of course you can do it, if your protein do not contain any structural S-S bonds othedrwise to protect the protein from one side, you destroid it from one other.

I do not think that ph=7 or pH=8 will change a lot in terms of DTT stability. The only way to prolong its life time is work on acidi PH e.g 5.5 were you will have the protonation of SH but in this case also your protein is subjected to this pH and whats acidic ph can cause to the protein depends protein to protein.

When in the past i worked with copper binding proteins where i need to mantain the Cisteines reduced to bind the Cu(I) the only way was to work in nitrogen atrmosphere after the reduction of the S-S. But to do this you need a glove box.

However generally a possible alternative strategy in the first purification step could be to replace the dialisys with protein concentration by ultrafiltration and rapid buffer exchange by desalting (using PD-10 coloumn that can load up to 2.5 ml of protein). In this way i was able to drastically reduce time and overcome the problems relative to the short stability of DTT.

good luck

Manuele

https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011306_TCEP_HCl_UG.pdf

Here you can find some information about the stability of TCEP at neutral pH.

Good luck!

Mahesh Chandak Maria Luisa Romero-Romero Thank you for the suggestions. i will work on it.

Manuele Martinelli Thank you for sharing your expertise in this regard. My protein of interest contains 7 cysteine residues. Initially i purified and concentrated upto 9mg/ml in the buffer 50mM tris pH 8.0,150mM nacl, 10% glycerol with 1mM DTT.

Right now i couldn't concentrate further above 1.8 to 2.3 mg/ml. protein precipitate in the PALL concentrator (10MWCO).

So i am thinking stability of DTT is playing some role in it.

Hi,

If you accidently left DTT at room temperature or at 4C for this prep, or did dialysis at 4C before and RT now then most likely DTT is the culprit.

Apart from the DTT issue

- Consider to reduce NaCl concentration.

- If adding edta dont have problem then you may consider using edta, and

some pmsf during dialysis.

- For concentrating use multiple of them. I always reused those concentrator to

concentrate same protein for 2/3 times succesfully as long as they were stored

in 20% ethanol. So, you may use 4 of them. It saves time as well as it saves protein.

- PI of the protein, and pH at which you are doing prep ( I am sure that you confirmed, and I dont think this would be problem for your prep)

Hi Mahesh,

The nature of DTT is keep on reduction of the SH group in your protein about only few days(recommendable amount up to 5mM). During dialysis it shouldn't affect your protein. Can I know how much you are using for dialysis?. TCEP about 1 or 2mM is more suitable for your work, Since it wouldn't affect the di-sulfide bond residues.

Mahesh Chandak

Hi,

-am adding raw DTT in the lysis and dialysis buffer at the time of prep only.

am doing overnight dialysis in 4 C, then go for concentration. That transfer time only my protein in room temperature.

In your previous answer you suggested to use 5fold DTT in dialysis then change desired DTT level at 1mM in the morning. In which method i can do it ? By dialysis or using concentrator(same as buffer exchange)

-Am adding 1mM pmsf and cocktail inhibitors in lysis stage only not thereafter.

-Thanks for the suggestion on using the multiple concentrator.

- PI is 5.76 including the his tag.

Manohar Radhakrishnan

Hi,

I am using 1mM DTT.

Hi Mahesh,

I have read that you use 1mM DTT. However your buffer is Tris-HCl and pH is 8.0. First, if you consider the pKa of the thiol groups of DTT (9.2 and 10.1), the reducing property at pH 8 would be very slow as only the negatively charged thiolate ( -S^-) is reactive and not the actual thiol (-SH) group. The propensity for the formation of oxidized dimers increases around that pH. That is why it is recommended to have 5-10 fold DTT in high pH buffers to overcome this effect. Second, if you compute the ions and their concentrations of your buffer components (all the ingredients of the recipe) at that pH and the effect of those ions on DTT, you can get the answer for your question how stable the DTT in your solution is. If your protein aggregates, then for sure you can know that either the DTT is unstable or the concentration of DTT is not enough in that recipe and pH. Better I would recommend you to replace DTT with TCEP (1mM to 5mM will be more than enough) in your buffer for better results.

0.1mM pmsf, and edta was safety suggestion because you informed that you are getting aggregation - (I can not say that adding them will stop it * for your conditions pmsf is only stable for 30 min or so at RT). You informed that you are doing overnight dialysis then 5 fold method you can do by the dialysis, and you can use amicon in the morning to change it to 1mM. Infact, dialysis would be fast to change it to 1mM - if you make like 2X buffer volume of what you used for overnight step- split in half and do it twice. I noticed that you are using very high conc prep - such preps have high chances to aggregate. As of now, I think if you do this dialysis and use couple of amicons - it will solve your problem. * you can mix protein from amicon gently after every 15 min of centrifuging. Let us know how it goes - Good luck !!!

Vinuchakkaravarthy Thangaraj

Thanks for the in depth explanation of 5 fold DTT usage.

will carry out the experiments with TCEP.

@Mahesh Chandak

Thanks for the detailed suggestion and good luck!