Hello,
I am trying to purify His-tag protein by affinity chromatography with Ni-NTA agarose but I am not able to elute it. I already purified recombinant protein with the same protocol few times without problems, but at certain point it just stopped working. I have tried to change the buffers and prepare everything from scratch but the protein seems to be stuck in Ni-NTA agarose ( as seen by WB). I also tried to increase the conc. of imidazole in elution buffer up to 500mM but nothing seems to work.
Any advice would be greatly appreciated!
Have you used EDTA to strip Nickel and then recharge it? Try to clean your column as thoroughly as possible (EDTA or NaOH or NaCl). How do you see that your protein is stucked on Ni-NTA agarose?
The problem might come from the expression of your protein in your vector and not the purification itself. I suggest you compare by SDS-PAGE an induced vs. non-induced lysate (right before you would put the lysate in your column) to see if your band of interest appears upon induction.
Also, purification issues often come from sonication (cells not lysed enough, or culture overheated during sonication). Have you checked if you loose most of your protein in the insoluble fraction/inclusion bodies ?
You said your protocol has already worked in the past. Was that with the same protein from the same E.coli clone (glycerol stock) ? If not, then it is "normal" to have to optimize some steps for each different protein you want to purify.
are you using the same used column? I think the problem could be due to storage condition of your agarose column
You need to give us some more info in order to help you.
How do you know the protein is stuck on the column? Did you take out some resin and run on SDS-PAGE?
Did you look for your protein in the flow-through?
How did you wash and elute? Imidazole concentrations?
Did you strip the Ni with EDTA?
It is not uncommon that similar proteins will elute at very different imidazole concentrations and this protein may need lower imidazole concentration to bind.
If the protein is indeed stuck on the column try eluting it with EDTA. If the protein does not come out with EDTA it is precipitated and you can try eluting it with Urea.
Albina Fejza Did you purify the same recombinant protein before using the column or is it a new protein? I had the same problem when I was trying to purify proteins from inclusion bodies. Could be due to improper folding of the proteins.
Hi Albina,
If possible, please show the SDS-PAGE gel results of the pellet, supernatant, Ni-NTA agarose, flow-through and elute after purification. According to your description, you suspect that the target protein is stuck in Ni-NTA. If it is confirmed, please treat the column with 6M GuHCl and recharge Ni-NTA resin.
Hai Albina Fejza,
Have you confirm the protein expression through SDS-PAGE? How the bands were appear. As per your description, wash the column thoroughly before protein purification and prepare the buffers and solutions freshly for purification. How do you know that the protein is stuck in the column itself. Is there any SDS gel images to confirm?
Jorgaq Pata I have loaded a bit of Ni-NTA resin in SDS gel to see if my protein is there after elution. And everytime I used new resin since I had these problem, so I didn't try to strip Nickel with EDTA
Martin Chenal Sorry, I wasn't clear enough! My protein is expressed by the cells and released in the conditioned medium( CM) so the purification is done from CM and not cell lysates.
Mohammad Moradi I don't know if I got your answer right.. I am using the same column but not the same Ni-NTA resin. Each time I wash the column and refill with new resin.
Bo Pontoppidan
Sorry for not giving all the details! Anyway,
As I answered also to Jorgaq Pata, I have loaded some of the resin in the SDS gel to see if the protein is still there and in the Flow- through we always have something left since we are not are not able to get everything out, but most of it is stuck in the resin. I was with 20 mM imidazole and I increased the concentration in elution from 250 mM to 500 mM. However, I also tried to elute with EDTA but it didn't work either. The only think I didn't try is eluting with Urea.. Thank you!
If your protein does not come off with 500 mM Imidazol it is probably not bound by interaction with the Ni-NTA. And this is confirmed by the inability of EDTA to elute it. Your protein has precipitated on the column and you need to find a way to keep it in solution (salt concentration, additives).
Albina Fejza your protein is soluble? It looks like it precipitates in the column, maybe because of the buffers, or maybe because of the high concentration in the column (rare but not impossible). So, you can try improving your buffer or load a smaller quantity to see if the problem persists or not.
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