While I have never tried refolding using IMAC, I can recommend the following:

• Balance your column with the starting urea buffer before loading the supernatant and make sure the buffer is compatible with your column.
• According to the literature, refolding is best using a declining gradient of urea at 4°C. If you are using a manual (gravity) column, you can prepare several buffers with declining urea concentrations for each step. see the following examples -

  Article On-column refolding of an insoluble histidine tag recombinan...

  Article On-column refolding purification and characterization of rec...

• the steps should generally be - equilibrate column, load supernatant, wash, refold using decreasing urea, wash without urea (optional, consult literature), elute, wash column. I highly recommend reading more articles to get a better grasp on the subject.

In theory, the histag binds to the column while the protein itself is a random polypeptide chain due to urea unfolding. As the urea decreases, the protein should fold by itself. The folding process at 4°C slows the kinetics and allows reaching more stable conformations, which are hopefully the correct ones.

1. yes, the column must be loaded with metal ions and equilibrated with adsorption buffer containing urea (+ a reducing agent if disulfide bonds can be formed)

2. equilibration and washing must contain urea, depending on the metal ions and chelating agent, the loading and washing buffer could be the same (+ reducing agent) or the second could contain some imidazole concentration to remove native proteins.

3. load (urea) - wash (urea) - refolding (decreasing urea concentration) - elution no urea + imidazole (all buffers must have a reducing agent).

As already mentioned, the urea concentration can be reduced linearly with a gradient, the time and temperature are critical. In general, long times are required to reduce the urea concentration to zero. PS, I have never been successful doing in-column refolding.

I have participated in such a procedure. The washed inclusion bodies were dissolved in 6M guanidine-HCl-containing buffer for several hours. This buffer also contained dithiothreitol to make sure the disulfide bonds were fully reduced, so it was important to use a Ni resin that is compatible with dithiothreitol. The solution was centrifuged, then loaded onto the Ni column that was equilibrated with the starting buffer, also containing 6 M guanidine. After a washing step, the guanidine-HCl was reduced to zero in a long, linear gradient. Both the start and end buffers also contained 1 M arginine-HCl to assist with refolding, and a redox buffer (a mixture of reduced and oxidized glutathione) to help form the disulfide bonds. Finally, the refolded protein was eluted with 0.5 M imidazole in the final buffer.

Most of the recovered protein was aggregated, so the last step was to run it over a gel filtration column to separate the monomeric protein from the aggregate.

Thank you everyone for the tips!

I will make sure to read thoroughly before I continue with this refolding steps

watch out for the reducing agent compatibility with your columns. You might want to take a look into cOmplete Hi-Tag columns or resin.

I have done my fair share of protein refolding. I haven't done it on a column what I do is denature my protein and then perform the affinity chromatography then take the fractions with my protein and perform dialysis using three buffers containing 4M, 1M, and 0M urea letting each go for at minimum 4 hrs. When doing the chromatography make sure your buffers contain the 8M urea to keep them denatured.

I think Michael Austin Pasquale might have a good point. Renaturation could be more efficacious after eluting the protein from the column.

Hi a little update:

I tried to do on column refolding like in the literature or as suggested but didn't work it. However I did do pellet wash before loading on the column with Triton X-100, 1 M NaCl, and regular buffer. I had no pressure problem as the lysate looks clear, but the refolding on the column did not really work out as there was no protein during the elution.

Next time I will try to purify it then do the protein refolding.

But thank you all for the suggestions! I find them very helpful

Adam B Shapiro - 1 M Arginine? When I have been using arginine I see a huge impact on binding capacity. Above 500 mM almost nothing binds for me.

My typical first approach for refolding on IMAC is dissolving washed inclusion bodies in 6 M Guanidine-HCl with DTT in case there are cysteines in the protein and adding 20 mM Imidazole. I don't use Guanidine in the running buffers, though. I start with 6 M Urea in the equilibration and washing buffer (adding 20 mM Imidazole by including 4% elution buffer on the ÄKTA). Then I do a gradient at reduced flowrate to buffer with no Urea - typically 10 CV at a flow of 1 CV/h and finally elute with a gradient to 500 mM Imidazole.

If the refolding works well this can give high purity since the inclusion bodies are often quite pure already and any other proteins which have metal affinity in their native conformation often don't when denatured.

Bo Pontoppidan Maybe it has to do with pH. I used arginine-HCl and adjusted the pH.

So do I. Interesting that it works for you.