I am doing IMAC protein purification with a his-tagged protein but I have been having issues purifying my protein.
First, the first few times I did it, I noticed that much of the protein was found in the flowthrough after pumping it through the column using a Gilson minipump. So I ran the flowthrough onto the column multiple times but still nothing being eluted as much.
Second, I thought maybe the protein that I am loading onto the column is too concentrated. So I diluted my protein sample by half and I got no bands anywhere on my SDS-PAGE. Not even in the flowthrough, not even in my eluted sample.
Third, I thought that maybe my sample is not getting eluted from my resin and is still bound. I took a sample of my resin and ran it on an SDS-PAGE but still nothing.
Where is my protein? Please help.
PS: I am also running westerns to probe against the his-tag to make sure it is his-tagged.
According to the information you provided:
First, it is possible that the location of your his-tag (N-terminus or C-terminus) does not allow this tag to be accessible to the metal ions on your resin (because of steric hindrance due to protein conformation). Try to change the location of your tag.
Second, It looks like your protein was proteolytically degraded during repeated loading and elution. Try to use commercial protease inhibitors cocktail at all stages of extraction/lysis/chromatography.
Third, since your resin after elution does not have your bound protein the probability of the first or second (or both, which is more likely) reasons are very possible.
It is a good idea to run western to probe against the his-tag. It would be better if that would have been done before the IMAC chromatography.
Hi Barney,
You are getting the protein in flowthrough, that means it is not binding to the resin. several factors affect the binding. Kindly check for the pH of the buffers( it should be either less or more than the PI of your protein). Imidazole concentrations should also be optimised. You can use 5-10mM in lysis buffer, 20-50mM in wash buffer, 250-300mM in the elution buffer.Dont include any chelating agents(EDTA) in your buffer, as it strips Ni ions from thr resin and maybe thats why your protein is not binding to the resin. Also, always use some protease inhibitors in your buffer so as to protect your protein from degradation in the cellular environment.
let me know if this is helpful.
good luck
Sometimes it can help to incubate your lysate with the beads for some time on a rolling wheel to increase binding time.
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