I used HisTrap to purify a His-MBP tagged protein, and then dialyzed the protein against 20mM Tris-HCl, 150mM NaCl, pH7.4. The protein is pretty soluble and I concentrated to around 10mg/ml, then the concentrated protein was subjected to Superdex 200 gel filtration column. However, the protein aggregated. I am wondering is there a method to disrupt the aggregated proein? Thanks~
Dear Gang,
To clean your column use 8M urea, with the temperature over 20deg, to avoid precipitation. This will wash your aggregate, but renaturation after this step will be rather hopeless...
Best - Jan
Dear Gang,
As Jan suggested you can clean your column with 8 M urea. You can also clean with 6 M guanidine hydrochloride. But whether you will be able to refold your protein or not depends upon your luck.You can try either amicon filtration or dialysis to remove the denaturant and then check for the refolded protein. But if some other protein is also stuck in the column then you would not get the pure protein. So you need to be careful if at all you want to recover your protein.
Best,
Jogender
It was not clear whether your protein was stuck on the column or not. If it eluted in an aggregated state, it may be possible to disaggregate it simply by changing the buffer. For example, it may require a high salt concentration or a different pH to prevent aggregation of the concentrated protein.
Try using sonication of low frequency. This might help in disrupting protein aggregates formed
In my experience, MBP fusions like 250 mM KCl instead of the 150 mM NaCl. With my fusions KCl gives no aggregate or I can use NaCl if I add divalent cations to the buffer (Ca, Mg) and obtain the same result.
Try applying mild sonication of the aggregate in phospahte buffer+salt. also think about treating the protein with edta overnight to remove unwanted nickel ions (etched from column) which can form bridges between the his-tags thus leading to aggregation. good luck!
Dear Gang,
I think it would be important to avoid that your protein aggregates in the first place, rather than trying to dissolve existing aggregates. So after your HisTrap you could subject your protein (still at a concentration
What is the PI of your protein ? Keep the sample couple of pH away from PI by using buffer during gel filtration. you may use ions, aminoacids, sugars and or surfactants like 0.01% polysorbate 80 to reduce or eliminate the aggregates
Hi Gang, are you using the same buffer ( 20mM Tris-HCl, 150mM NaCl, pH7.4) on the Superdex 200 column?
Your protein (at 10mg/ml) is soluble in this buffer, right?
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