His-tag and Ni-NTA purification for my 18kDa protein hasn't been very successful. I'm using 10mM imidazole in binding and 25mM imidazole in wash buffer. Still, I see a lot of non-specific protein eluting although i changed the old resin to new. Thus, i wanted to get rid of non-specific proteins that are above 20kDa from the lysate and then do affinity chromatography. Ammonium sulfate seemed an obvious approach. First i tried 45% to 75% salt saturation in sodium phosphate buffer to see if my protein is precipitating at higher saturation concentrations, since % salt saturation and molecular weight of protein are indirectly proportional. I corrected the pH from 6.0 to 8.0 after 2 hours of incubation so that all salt is solubilized. However, i saw most of my protein, concentrate and precipitate at 45% when i was expecting it at 50% - 75%. So, i tried 10% to 60% salt saturation at 4c and 8 hour incubation on rotation. I find my protein concentrate at ~ 40% mostly. However, main problem is rest of the proteins regardless of molecular weight are also precipitated at the said saturation. What can i do?
First, you need to recognize that IMAC is a pseudo-affinity technique. You have to modify conditions such that the selectivity for your protein increases (increase initial imidazole concentration, switch metal ion, etc.). You can also run an imidazole gradient for desorption.
Second, protein precipitation by ammonium sulfate depends on the hydrophobicity of the protein surface.
Ammonium sulfate fractionation as the first step can be useful if you get some enrichment and a decent yield.
To separate proteins on the basis of molecular weight, use gel filtration chromatography.
If you are having trouble purifying your protein by a single step Ni-affinity chromatography (not uncommon), the solution is to add additional chromatography steps that operate on different principles, including gel filtration, ion exchange, and hydrophobic interaction.
@Adam B Shapiro, Thank you for the response. I am also using SEC post his-tag purification. However, since the his-tag elutions are very dirty, it was very difficult to extract my protein even in SEC. Which is why i
m trying to get rid of proteins that are not supposed to bind to ni-nta resin.
@Omar Gonzalez-Ortega Thank you for the response. I've tried to increase imidazole concentration during binding and washing and it too didn't work very well.
Run an imidazole gradient for desorption. Swith metal ion to cobalt or zinc.
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