Sorry, but I'm going to answer with a question. We found that a protein we're purifying likes ~400 mM KCl and precipitates in less. This salt concentration will work OK for IMAC, the first step in the purification, but I'd predict it will be incompatible with ion exchange chromatography (which is our typical second step). Any suggestions for subsequent chromatography steps that tolerate high salt? We already have DTT and glycerol in the mix. Thanks.
Gel filtration, of course. Also, sometimes dilute proteins may be stable with low salt. You may dilute the protein just before ion-exchange, load the diluted protein on your ion-exchange column and then elute with high salt. DTT will effect IMAC but not the other methods.
thanks for the suggestions. it's already pretty dilute, so we didn't want to use an even more diluting method (gel filtration). I also hate the idea of proteins aggregating within a GF column. but maybe fast dilution + ion exchange can work.
You can also try HIC with ammonium sulfate reverse gradient for elution. This kind of intermediate purification step is as suitable as IEX after IMAC capture...If your operating conditions are well chosen you will even be able to concentrate your protein. If you need to concentrate your protein without changing its environment, tangential filtration might be an interesting option.
HIC requires elution with low salt, so back to square one as the protein will then precipitate.
What's tangential filtration?
@Bhupesh: thank you for commenting back !
1- Yes, you're right it's a risk...But as long as you don't know the ammonium sulfate concentration required for protein desorption you can't tell. Some proteins are adsorbed at 5-6 M ammonium sulfate and desorbed at 0.5-1 M...It is just hypothetical, it will depend on protein hydrophobicity, stationary phase polarity,...
2- Ultrafiltration through microporous membranes. With an appropriate membrane cut-off, proteins can be concentrated (without loss if they are totally retained by the membrane) without changing buffer composition (if microsolutes composing it aren't retained).
Maybe,First,you should be sure the reason of aggregation protein。sometimes, the reason is not for high protein concentration。High concentration may cause random aggregation。you can confirm it through Size exclusion。in my opinion, aggregation more as incorrect folding. you can try add chaperon 。
Hai Bhupesh Taneja , the Tangential Flow filtration (TFF) one kind of ultrafiltration using with membrane with opt cut off protein value, like 10,30 100 KDa pore size, this type of techniques maximum using in the downstream preocessing of the vaccine production.
This techniques used for the concentration of the protein, as well as the removal of elution buffers like NaCl , etc
thanks you Xia Chen, Actually our material was Affinity purified eluted with different molar concentration of NaCl,, along with protein, furthermore we are focusing on the removal of salt by TFF method, during that techniques it was aggregated
Reduce the salt concentration when your are purifying the protein and dialyse the elution fraction to remove the salts .
Before storing your protein add 10% glycerol in the buffer.
I am afraid the ultrafiltration (Mricropore) through membranes will not be much of a help. Should the protein naturally an aggregate, it is going to aggregate anyway. I suggest that adding a anti-chaotropic agent while ultrafiltering may prevent aggregation, use with caution, it may denature the protein. I used Micropore on soluble proteins with excellent results, it is rapid and quantitative to remove salts..
"Effects of chaotropic and antichaotropic agents on elution ofpoliovirus adsorbed on membrane filters". PNAS, 78, 1229, 1981.
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