Dear all,
I am currently purifying a small protein which contains 5 cysteines. The purification flowis: Ni-NTA -> His tag removal by TEV cleavage -> remove TEV protease by Ni-NTA column -> phenyl sepharose.
20 mM beta-mercaptoethanol (BME) was added to all buffers, including lysis buffer. Then, after removing TEV protease by Ni-NTA column, BME concentration was adjusted to 50 mM prior to phenyl sepharose column.
The buffers for phenyl sepharose purification all contained 50 mM BME. The figure attached are results from phenyl sepharose purification, which 2 bands are obviously present, indicated by the red arrows. However, after leaving the fractions at 4℃ overnight the band shifted downwards, which is desirable. We expect that the lower and upper bands are the reduced and oxidized form of the protein, respectively. (I only want the reduced form).
The problem is, this approach is not very reproducible. Sometimes the bands shift downwards and sometimes they don't. I would be very welcome for any suggestions.
P.S. I am sure the upper/lower bands are not monomeric/dimeric states of the protein as the size do not double. The proteins are run in 16% tricine SDS-PAGE.
Hello Pongpawan,
you can use 0.5 mM TCEP in all your buffers, that would keep the reducing environment for your protein and not interfere with Ni-NTA.
You can also run reducing and non-reducing SDS-PAGE to compare. The only difference in the presence/absence of DTT in the loading buffer.
If you protein needs however the correct formation of S-S bonds, then it you can try the Origami strain of E.coli or mildly oxidize your protein in vitro.
It's not obvious to me that the two bands are caused by oxidation & reduction. With your massive BME concentration, you should not see any trace of an oxidized band. Proteins are produced in a reduced state in the E. coli cytoplasm. There's nowhere those electrons could go to. More likely, the two band are caused by proteolytic damage of some sort. Try a protease inhibitor tablet to prevent it.
@john I also agree that theoretically, there should not be any oxidation due to massive BME conc. However, for the two bands, I don't really think they are proteolytic damage as the lower band (the expected reduced form) has the expected size.
if you have an antibody to the protein then perform a western blot to see if you are getting proteolysis on your sample. you should have a protease inhibitor cocktail in the mix. If your protein has to be in a reduced state then using his-tag really isn't the best method of purification.
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