Hello, everyone! I have seen many protocols for protein purification that use DTT or BME. I understand that it is to "break" disulfide bonds, but I would like to know what this implies in terms of contaminating proteins.
I am asking this because I am purifying a protein for which I have tried all possible protocols and it always comes out full of contaminating proteins (a lot of them)... Could the use of DTT help with this?
Dear Bruna Ukrainski
DTT may prevent aspecific protein aggregation in case that proteins contains free cisteines exposed in its surface, but at the same time it can even promote protein unfolding for those protiens containing structural disulfide bonds. Everything is possible and it depends case by case but i do not think that DTT may help to reduce contaminants. optimization of purificazion process, ionic strenght of buffers, speed of gradienta, may affect.
good luck
Manuele
It sounds like the problem is with the purification process, as DTT or BME will not necessarily remove contaminants. Describing your protocol in greater detail could help us identify potential problems.
In general, you should consider using a high resolution size exclusion column to eliminate junk protein. Best of luck!
I have purified using several conditions, but in general I use 20 mM HEPES, 500 mM NaCl, 5% glycerol and 500 mM imidazole in the elution buffer.
I have tried using low concentrations of imidazole in the wash buffer, but it completely removed my protein from the column. I have also tried using the cognate amino acid of my enzyme (I work with an aminoacyl tRNA synthetase), with MgCl2, triton x 100, varying buffers for TRIS and phosphate... in short... nothing works.
When I tried an ion exchange my protein "disappeared", it was not in any collected fraction. It is very difficult.
Regarding nickel affinity columns:
- If low imidazole removes the protein, that may indicate a problem with the Histag. Some imidazole in the lysate (10mM) may remove non-specific interactions.
- The position of the Histag may also be crucial (N\C terminus) - sometimes its on the wrong end of the protein. Unfortunately, these cases are difficult to determine.
- Make sure the Histag exists on the DNA level, just to be safe.
- What is the pH? This type of columns require a specific pH range to function. Be sure to consider your protein's PI as well to avoid aggregation.
Regarding anion exchange - check the PI of the protein versus the pH you use to purify it. If your protein has a positive charge, it would simply pass through with the rest of the lysate. Try to optimize the pH to be 1 unit above the protein's PI, if possible.
Also, there is a chance that you get inclusion bodies and very little soluble protein. Be sure to use a rich media (if possible) and long induction times at RT (for E. coli).
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