Hello, I'm working on purifying human recombinant protein in an E.coli system.
After several purification steps, I analyzed purified proteins with the Waters HPLC with SEC columns, but my protein showed different elution time only in HPLC SEC columns, and I want to know possible reasons about it.
My protein is 23 kDa, and its PI is about 9.6. I used PBS buffer with pH 7.4.
There was no aggregation, the size of protein was viewed with SDS-PAGE, and size was same with control commercial protein (expressed in Cho cells).
I used proper assay kit to test protein's activity, and it worked similarly with commercial protein.
I used HPLC SEC columns, TOSCH, TSKgel G3000SWXL and Waters, BioSuite 250, 4um, UHR SEC. Both of them can be used in analysis about 10-500 kDa proteins.
Also I checked column's purification range with a standard protein kit, 14, 29, 44 kDa proteins, and there was no problems in columns.
I loaded my purified protein, but it eluted after 14 kDa size.
Commercial protein eluted between 14 and 29 kDa size.
Then I used Gel filtration column (HiLoad 16/600 Superdex 200pg) on Akta avant, but it eluted between 14 and 29 kDa proteins, different with HPLC SEC.
These results showed only in HPLC SEC columns, my protein eluted strangely.
I don't know how could this happen.
I hope someone knows these problems.
Hello Jungon,
It seems that your protein interacts with the matrix of the TSK gel, which results in retardation and elution that would correspond to a lower Mw than expected. This does not occur with the Superdex column, which uses a different matrix.
Pierre Béguin
Thank you for the possible information.
It was an odd situation, but I think it could be.
Thank you sincerely.
It indicates SEC column has a lagging effect on 23 kDa protein and Supersex gel filtration column shall be preferred. Secondly, use Phosphate buffer of pH 8.5 that shall create a low positive charge on 23 kDa expressed protein which this may resolved better on SEC column, too. Just try!
Also maintain a uniform flow rate of mobile phase during hplc analysis.
Shamsher S. Kanwar, thank you for advises.
The columns recommend using ph range between 2.5-7.5, but it can be possible.
My protein has his-tag, not in commercial protein.
Therefore, I decide cleaving his tag first and watch for difference.
Thank you, again.
I found addition of 0.2 M arginine hydrochloride can solve this problem, and it perfectly worked for me, testing several recombinant proteins.
I hope anyone, who might have similar problems future, could be helpful with this paper.
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