I am following an established protocol where during initial Ni-NTA purification the solubilised pr solution (6M GuHCl/0.01M Tris/0.1 M NaH2PO4, pH 8) is incubated with Ni-activated chelating sepharose beads for 2 hours and then flow through is collected using column housing. This flow through is then again incubated with second Ni-beads o/n and, similarly using column housing Pr is eluted from the bound beads with decreasing pH of the same buffer.
Now, during collecting 1st flow through I pushed it with high pressure air from syringe-piston to get the flow through quickly and I found the 1st adsorbed beads became white. I found very less amount of protein after elution from 2nd adsorption. I suspect that Ni was forced to go out of the beads so as His-tagged Pr with it.
Did you perform a test for presence of Ni in 1st and 2nd flow through? You need to optimize your elution buffer. Did you try to check different conditions for your elution buffer?
What is the immobilized chelating agent in the adsorbent? What is the low pH buffer? Was this result observed before using the same column and conditions?
Hi Alemu,
Thanks for your query. No, I have not. How can I do that. I am just following an established optimised protocol. I have done this purification before. This time I hurried. The first adsorption we normally discard. From the second adsorption we collect the protein. If I run a gel will that help? How should I run as that is in 6M GuHCl buffer. What if I add some fresh unactivated washed beads directly and incubate it o/n will that Ni (if present) binds to the bead?
Hi Omar,
Thank you. I used chelating sepharose beads that were activated by Ni, PBS and 6MGuHCl/0.01M tris/0.1M NaH2PO4 pH8 buffer. The result was normal before. I saw the first adsorption cloudy normally (light bllues is the fresh beads color) . The second adsortion after o/n incubation is aslo little bit cloudy but less than 1st. We wash it with same buffer 6MGuHCl with decreasing pH and eluate it at pH 4.5
Well, nickel ions can only be displaced by other stronger chelating agents or a low pH. Is it possible that the sample you are processing contains something like edta or the like. Also, you can expect some ions to be stripped when protein adsorption occurs. Nonetheless most of the ions are not accessible to protein molecules.
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