I'm trying to purify the His-tag protein recently, but no matter what have I tried, there is still precipitation when I want to concentrate the protein after His-tag column. The solution was clear before I concentrate it, but when I wanted to concentrate it, problems came out.
I was using 500mM IMDZ to elute the protein, and the solution became cloudy and also some precipitation came out when I concentrate it. Then I tried to add some DTT in the elute solution, this time, the sample is not cloudy, but still have precipitation, and the sample became a little yelowish. I even tried to add 1mM EDTA after the elution to bind the released Ni, but there seems no difference. If I centrifuge the sample and use the supernatant, the amount is not enough.
So can anyone give me some advise to avoid the precipitation and yelowish when I concentrate the sample solution after His-tag column?
Hi,
Maybe the saltconcentration is to high after concentration.
You could try dialysis prior to concentration.
With kind regards,
Ronald
Hello,
I guess your protein requires ions for stability. You haven't mentioned
your protein, therefore I can't tell for sure but i suggest try to add different concentration of appropriate ions.
Lets say, your protein is a Beta-lactamases, so add Zinc or if it is a Ni transporter, then add Ni ions. If you have already fulfilled these requirements, then I will also go for Ronald suggestions. Further, you can test your protein with different concentration of NaCl in your buffer...lets say, 50, 100, 150 and 200. Good Luck
@an hour ago Ronald Van Vlierberghe
Thank you for your answer, sorry I didn't mention the concentration of the salt of elute buffer, it was 500mM NaCl. And as I know, under a limit, isn't that higher concentration of salt could make protein more soluble? There is a difficulty that my protein can not stay with IMDZ too long, last time I tried dialysis, and it became cloudy even before I concentrate it. I'm thinking buffer change column.
@Ashfaq Ahmad
Thank you also, but my protein does not need any ion except Mg+, we even added EDTA to bind the metal ion before.
Hello,
can you lower the IMDZ concentration for the elution? It can be, that your protein doesn't like IMDZ or high salt in general (as already mentioned above). You can use 200 mM NaCl in elution buffer and lower IMDZ. If you can't use lower IMDZ with your column, use for example talon resins/column - 200mM IMDZ in elution buffer (and 5-10mM in binding/wash buffer) works nicely. You could probably use even lower concentration of IMDZ for elution with this column.
Finger crossed
@Katerina Zabrady
Thank you very much for your advice, this is very helpful acturally I will try it today!
Note about salt concentration and solubility: With higher salt the iont interactions are weaker, but the hydrophobic interactions are stronger. That is why the high salt can cause insolubility especially in case of proteins with highly hydrophobic surface.
I agree, play on salt concentrations.
Also what is the pH of your buffer and the theoretical pI of your protein?
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