I performed NTA-column chromatography (Hi-Trap-His-Column, 5 ml) with loading under denaturing conditions and did on-column refolding. I elute with 800 mM imidazole and cleave the tag with thrombin protease.
Before performing my activity assay, I need to get rid of the imidazole and the thrombin and change the buffer to 8 M urea and pH 2. I want to achieve these two tasks by gel filtration.
I now have the problem of choosing an appropriate column and would appreciate some input here :) Don't know which column material can stand pH 2, for example. The column should run as fast as possible and the peaks should be as narrow as possible, but I think this depends more on the flow speed, than on the column material.
My sample volume is 5 to 10 ml (which I would like to apply without concentrating the sample before loading - if possible).
Thanks in advance for any help here :)
Almost impossible to get what you are asking for. Let me explain. If you are looking for high velocity of the flow through the column, than you will have to sacrifice resolution. Highly accurate columns (Superdex 75, 200, Superose 6) have more stringent limitations as to the flow speed and input volume. Less accurate gel-filtration columns (Sephadex, Sephacryl, Sepharose CL 6B) may allow you to use higher flow velocity but the resulting peaks will lose their sharpness. Again, depending on the column size, you can not load more than a certain amount of total protein (column documentation gives you the exact number). Otherwise you loose accurate separation.
Next, ideally, the size-exclusion chromatography is done in the buffers that are identical or very similar to the samples buffer. That can be omitted only in the cases when the sample volume is significantly lower that the bed column volume. So, you are either going to do multiple runs or you risk to loose resolution. As you are going to substitute GuHCl with 8M urea, I would do dialysis first, and then applied my dialyzed samples onto the gel-filtration column. All the columns I have named above can be used with high GuHCl or urea buffers. Virtually all of them can be used for a short period of time (like for column cleaning runs) with buffers that have a low pH (2.0).
So, do your runs, and then re-equilibrate the column with the buffer that has a neutral pH. For resin/columns details, see Amersham Bioscience site/gel filtration.
Good luck.
Hi Peter,
You will be hard pressed to find a GFC column which can withstand a pH this low... the closest thing we offer has a pH range of 2.5 to 7.5 (which is close). This is the BioSep SEC column. If you can adjust your pH upwards to 2.5, this column should work well for you.
Paul
Peter,
First of all, you refold your protein on the affinity column and then you want to add 8M Urea again? Is that correct?
Then I may suggest you simply leave the 8M Urea and elute with pH 2 buffer. If your protein elutes at 800mM imidazole you can wash the column with 500mM and change back to 0mM before elution. Your product should be pure.
If you really need to worry about the tag, elute with standard buffers (no Urea), add affinity tagged thrombin and run it over a standard column again to get rid of the thrombin.
You then may change the buffer with a Sephadex G-25 desalting column, which should be stable at 0.1M HCL according to the manufacturer. If your protein is pure after the Nickle column (and supposed to be denatured anyway) you don't need to worry about clean peaks.
Hi Peter,
please pay attention to the remark of Stefan: using of 8M urea after refolding is puzzling. I am a bit sceptical about gel filtration because we do not know the molecular weight of your protein, which may be similar to thrombin and thus their peaks overlap during separation on Sephadex. I would think of using immobilized thrombin for cleaving the tag. You may then remove imidazole by dialysis against a buffer with pH 2.
Alexander
Thanks for your input! I need my protein in 8 M urea for my functional assay (fibrillation). I would definitely be eluting directly with buffer containing 8 M urea, but the problem is the tag... For thrombin cleavage to occur, I must elute the protein in native buffer :/ cleave the tag and then denature it again...
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