I want to isolate a 25kDa-protein, but impure proteins are different in size within 10kDa. how can we tell whether the SEC column could separate proteins of interest from impure ones? I attach the gel electrophoresis result after SEC. Thx a lot!
Hi,
Superdex 75 has a higher resolution than Superdex 200. If your target protein with high concentration, you can also try a long gel filtration column, depending on the characters of your protein.
Hope it helps!
Use Superdex 25 to first separate proteins of 5KDa from your protein of 25 KDa and then subsequently use long column od Superdex 75.
Hey! I would try a spin filtration (like 10MWCO) to separate 10kDa impurities before loading on the size exclusion column (I don’t know you are already doing this or not). After spin filtration you can run gel again to make sure impurities are still there or not. So I think steps like, spin filter - run gel- Q column - spin filter again - run SEC - run gel, might help. Best of luck!
Actually, your SDS-PAGE shows that the SEC is working beautifully, proteins with higher molecular mass leave the column first, followed by those with lower mass.
You should repeat the electrophoresis with a ladder of proteins of known mass, then you know in which fractions you can find your protein of interest. Personally, I like the "rainbow markers" from GE, because each protein has a different colour, making it easier to tell which is which. Given the size of your target protein, the low molecular mass markers are appropriate.
The gel also shows the limitation of SEC: it is difficult to get baseline separation of proteins with similar size, and proteins of different size if they bind to each other. You will have to combine SEC with some other method, preferably one that allows gradient elution (IEC, HIC). Usually, SEC is done last, using a small sample volume and a long column.
Thank you, everyone! So there is no "absolute" resolution in SEC but we could refer to the manual to see the relative ones. And a longer column has a higher resolution than the shorter one. I think above answers are really useful in terms of my original question.
However, the problem is that the protein degraded in the process of purification. I recalled the process as His-tag purification-spin filtration-SEC (Superdex 75)-gel... Perhaps my problem is to figure out how to stabilize the proteins in the first place.
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