It actually is not always a good thing. DTT will reduce the disulfide bonds, which in some cases causes causes irreparable loss of structure and activity to what you are trying to purify. However, if your protein does not have any disulfide bonds, if it is thermodynamically stable and always comes back to its native structure after being denatured, or if the disulfide bonds are so protected that DTT is not powerful enough to reduce them, then you will get some benefit out of DTT. The DTT can, for example, inhibit the activity of certain proteases which are rendered inoperable upon loss of their disulfide bonds. DTT can also separate your protein from some other proteins that are adhering via indiscriminate disulfides from surface interactions. It So, I wouldn't always use DTT in a purification, but I often will.

If you need to get rid of DTT after a certain step in a purification, you can always dialyze it away. If that doesn't cause your protein to come out of solution, it will do a good job of prepping it for your column.

DTT usually use to reduce the disulfide bonds in the protein structure, and protect from oxidation stress.

For your experiments, I assume you never try to detect by Western after column purification. If so, let's try to purify the target protein in small scale, and please check the protein by Western. Your protein do not mind oxidation, you can get the protein by purification.

Another suggestion, please try western the following sample; 1) normal extraction (with DTT), positive control, 2) without DDT extraction, negative control, 3) protein extract without DTT and add DTT before western, 4) dialyzed sample (mention the above Lasseter) after normal extraction.

Is it worth considering an alternative reducing agent after youve ?extracted (or which might also work for the extraction). Clearly mercaptoethanol is not compatible and it may be that all reducing agents are incompatible. But there are alternatives that might work such as TCEP which has no thiols but does break disulphide bonds

Thank all for your valuable comments.

Dr. Daisuke Kobayashi I will try to follow your suggestions. I wish it will work. And yes, I did not run WB after column chromatography as i could not find proper extraction buffer so column chromatography was not done.

Dr Mary Phillips-Jones , though the use of TCEP prohibition is not mentioned directly in the datasheet, but TCEP also breaks disulfide bonds. So may be it is also not to be used.

I routinely use 1mM DTT or TCEP in my Flag purifications.

I think what happen is aggregation of your protein due to S-S linkage since DTT is a reducing agent and usually used as proteases inhibitors .You can check your gel and if there is aggregation of the protein as i suggest you can see the aggregation will not enter the gel and stay at the application point.Good luck

I am agree with Dr. Kobayashi. You can try with both positive ( with dtt) and negative (without dtt) and check both SDS-PAGE as well as western blot. Hope you will good result!