A recombinant his tagged protein (Size: 27 KDa Theoretical pI: 7.85) is eluted fine with 50 mM NaH2PO4 pH 8.0, 500 mM NaCl and 250 mM Imidazole. Precipitation occurs during dialysis with same buffer minus imidazole. Is the precipitation due to proximity of buffer pH and protein pI? If so, should I use a different buffer for dialysis or for both elution and dialysis? Is 0.1 M sodium phosphate buffer (pH 6.5) a better candidate with pH being one pI point away than protein’s? The MWCO of dialysis membrane is 14KDa and the protein is purified for antibody production.
Nagganatha Suganthan Ravi Hi, is your protein isolated from inclusion bodies?
What could be happening is that your protein is denatured either before or after the IMAC step. Imidazole might be keeping the protein soluble because it has hydrophobic properties.
Dear Nagganatha
i think that the proxymity of ph to the pi of the protein can be a possible driving force that induce aggregation.
generally imac work better at ph around 8 because the histidines at this ph bind better to the metal but you can certanilly try phosphate ph6.5 as well as use tris instead phosphate and increase a little the pH of your buffer eg 8.5.
however this is not the only factor that can induce aggregation for example:
1) Did your protein contains cysteines? in case try to resuspend the precipitate and load it in sds page with and with out reducing agent to see if during the dialysis you have aspecific intramolecular s-s formation that result in aggregation and precipitation.
2) did you protein contains a metal binding site? because in this case there is also the possitimite that it was able to extract some nickel from coloumn which crosslink beetween protein molecules and promote aggregation. in this case you can add some edta in dialysis buffer and see if inhibit from aggregation.
3) Is it possible that during dialysis the protein was degraded from some impurities? did you run sds page of the precipitate ?
unfortunatelly there is not a Universal solution valid for all the proteins.
in and case, i suggest to yuo to replace dialysis with desalting which is faster
you can found informations about it on my blog: https://proteocool.blogspot.com/?m=1
ProteoCool 8: buffer exchange methods overwiev and ProteoCool 11: Rapid buffer exchange by PD-10.
pd-10 can seems expensive but yuo can re-use a single coloumn many times.
Good luck
Manuele
There is no rule saying that you can not change buffer during and after purification. precipation could be very well due to ph being too close to pi. there is no harm in using ph 6.5 but ph 7.0 might be ok and its more physiological. There is one problem in going from ph 8.0 to 6.5. Your protein is going to see ph around pi for quite some time which can cause precipitation.
you can try NiNTA at ph 7.5 and then dialyze your protein in pH6.5 buffer. Another option. Do your purification at pH8.0 but elution at lower ph buffer like ph close to 7.
also adding edta in your elution fraction before dialysis might helps. If its not metallo protein, I almost always add it. Ni often leach out of matrix and can cause precipitation.
Dhiraj
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