Hi there,

Similarly to what may happen on NiNTA column, it might be that your protein is binding Ni2+ ion and DTT reduces it giving the brown color. Maybe the concentration you actually had in your dialysis is higher than previously.

Hi.

Someone from our lab faced that issue too..but I am not sure what happened after that. Although I have not personally faced that problem, I feel that the issue does not arise from the powdered DTT (unless it is contaminated with another component). It could be related to the batch of protein that you were dialysing (unless it is the same batch of protein that you used previously)

You might have added too much DTT

Hi Yong Jia,

I added DTT to get its final concentration of 1mM, in buffer.

Have you double checked it? one experience I got with protein purification is that, the color of the protein solution tends to darken if you add too much reducing agent. But not one hundred percent sure with your case, just a factor you may want to check.

ur  protein has some co-factor or ligand, may be do AFM you may be able to get some very useful results.

As Rajagopal Kammara said, your protein might bind some type of co-factor under reducing conditions. I once had a protein that purified as a brown color, which disappeared after a day or two. However, if I then added DTT, some of the brown color would come back. That protein turned out to be reversibly bound to iron. Using basic spectroscopy, an iron-sulfur cluster in a protein should provide a 'shoulder peak' off of the 280nm peak... around 400-440nm (depending on what type of iron-sulfur cluster it is). Also - if I put TCEP (instead of DTT) the solution turned 'pink'.  Try dialyzing the protein with 1mM Fe2+ and 10 mM DTT overnight. If the solution turns even more brown, (and you see a 400-ish shoulder peak) then you can begin to have some confidence that you are working with an iron-binding protein. If that is the case, consider yourself very lucky, since many Fe-S cluster proteins are very hard to work with and often degrade or denature upon exposure to oxygen. 

In presence of DTT , protein attains denaturation state, where its surface is exposed hence free ion containing mol. Such as Fe2+ will bind to them. Therefore, the protein turns into brown color. Try to oxidize the protein it will lose its brown color. These stories r very interesting to study n fetch high impact factor publications. Further to understand AFM will give perfect idea how many mol of Fe is binding etc., I hope this info is satisfied ur queries.

Thank u all giving me such valuable info.