Hello everyone,
I am having trouble isolating and purifying an extracellular protein which I have expressed in E.coli. I have induced my protein at 0.5 mM IPTG by incubating it at 37 degrees for 6 hrs which gives a thick sharp band when visulaized on SDS PAGE. However, when I am trying to purify it using Nickle NTA-Agarose, No protein is visible on SDS PAGE. Please give me some suggestion on how to purify the extracellular protein expressed in E.coli.
Dear Dr Krishna Rai you can use Ammonium Sulphate Precipitation method, and can also concentrate your solution by vacuum evaporator or by water bath depending on the nature of the protein. Use online Ammonium Sulphate Calculator for saturation.
I think this will be helpful for you and can contact with me I have already done it.
Best of luck in advance.
The only way to purify extra cellular protein is as Ram Krishna suggestion.
But if you want high purity protein after precipitation you try Ni-NTA agarose or column chromatography by size or ion exchange chromatography.
But all these depends upon the nature of your protein.
So initially you standardize these parameters with small volume then go for larger.
Normally, you should be able to fish out your extracellular protein by passing the culture supernatant over the Ni-NTA column, provided that the conditions are right (no EDTA, pH ≥ 7.5, imidazole < 20 mM). If not, it could be that the protein you are seeing has been proteolyzed and no longer contains the His-tag. In thi case, you will have to use generic purification techniques, such as ion exchange or gel filtration chromatography
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