I have used two different His-tag resins in batch mode to purify at His-tagged protein. The protein is bound to the resin in 6M GuHCL pH 7.9, and eluted with 500 mM imidazole pH 7.4, with both resins most of my protein has been staying on the resin after the batch elution steps. Has anyone run into this problem and found a solution? Would like to avoid using a low pH elution. I have 5% glycerol and 300 mM NaCl in the elution buffer, thinking of adding 5% glycerol to the binding buffer since the protein may be binding to the resin via a hydrophobic interaction.
Is it possible that the contact time between the resin and the elution buffer is too short? Are you using a drip column, or just mixing the resin with the buffer? I would suggest to use gravity column, and provide ~ 5 min elution time per ml of the resin volume.
Hi Sanjay,
The resin was batch washed with elution buffer 3X, and I can see a elution curve on the gel, so it is being washed off the resin, but the resin still has a lot of protein bound to it after the third elution wash. I have also run into the same problem using a FPLC and column with extensive washing. That is why I am now trouble shooting using the batch method on small samples of lysate. Have very good expression levels in E. coli, but do not want to lose a large amount of the protein on the column since I need to purify it on a second column.
Probably then the unfolded protein is aggregating / non-selectively binding the resin. You can confirm the ionic-binding by stripping the His-Ni interaction with EDTA. If the protein is selectively bound, it should elute with EDTA. You could add low concentration of Triton in the binding & wash buffers.
Try longer incubation time for elution. I usually let the second elution volume sit on the resin for ~15-30 min.
Otherwise, your protein might be aggregated on the resin. Try higher salt concentration or different pH. As you probably know, imidazole is a weak buffer and it will affect the pH at the concentrations you're using, pushing the pH towards 7, where your protein might be insoluble.
If the protein insnt coming off with 500mM imidazole then you have a real problem with the protein and what you have made is bad. You mention the protein is loaded with 6M GuHCL are you reducing the GuHCL at elution? If so the protein is probably aggrigated and suck to the resin, especially as you mention you the protein may suffer from hydrophobic interactions. I have seen this with the type of protein we work with (membrane proteins) we have purified GFP-labeled proteins which upon elution the column still stays green, even with a EDTA strip and NaOH washes. If its a hydrophobic interaction try washing the column (on the bench at RT) with imiazole and some SDS if the protein comes off then this will let you know that the protein has ppt out on the column
It probably precipitated on the column as you eluted directly with buffer containing no GuHCl. As others have said, the protein is not folded and refolding may not occur on the column in the buffer systems you have chosen. To refold on a column, one usually slowly decreases the GuHCL concentration with consecutive washes. But this does not always work, and if it does not, your protein will not only aggregate, but come out of solution or precipitate.
let me know if you are using NTA or IDA as a complexing agent , NTA hold the protein more strongly than IDA , IDA have other benefits like more ligand density. Seconldly role of metal ion i hope you are working with Ni2+, the binding can be more strong if you use Zn2+/Co2+.
Thank you everyone for the feedback,
The His-tagged protein binds to resins with Ni and Co metals (tried two different resins). I think the protein binds to the resin through hydrophobic interactions when it is in 6M GuHCl. I just need to have a high His-tagged protein-to-resin ratio to first saturate the hydrophobic sites on the resin and then the remaining His-tagged protein binds to the cobalt on the Talon resin. Then I can get plenty of re-folded His-tagged protein to elute from the resin with 150 mM imidazole. I do a gradual refolding on the column by reducing the GuHCl concentration in the presence of 300 mM NaCl.
I think this is a common problem with IMAC resins, but who regularly checks the resin after elution for bound protein, especially when you get a His-tagged protein elution peak? (however small). With pre-packed columns you would have to break the column apart to get the resin out for analysis. And it was not just the His-tagged protein that bound to the resin via hydrophobic interactions, plenty of other E. coli proteins did too, but they were not over-expressed so the individual E. coli protein bands were not as prominent as the His-tagged protein (~30-50 E. coli proteins were bound to the resin).
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