Hello everyone,
I have been trying to express and purify a protein. I am getting a good protein yield after Ni-NTA chromatography, as seen in SDS-PAGE. Problem is after purification, during dialysis it got precipitated( dialysis was done against deionized water). I diluted the precipitate many folds but it did not solublize. Again I tried to directly dilute the elutes after purification, but to my disappointment it again got precipitated. I further dissoved the precipitate in 4M urea but its not getting dissolved. I am not getting any clue, to what should I do. How to move ahead and desalt the protein and purify it further. Kindly help.
I am attaching the gel pic of protein after Ni-NTA colun . It doesnt contain protein marker. Also, along with the main purified protien other protein bands are also coming, albiet running a Ni-NTA column. Why so? any suggestions for this problem?
Check the precipitated protein for proteolysis by SDS-PAGE. One possibility is that it became insoluble because of proteolysis.
Try changing the pH and salt type and concentration.
Try including a ligand, if there is a low-molecular weight ligand that is readily available.
Change the temperature. If you are keeping the protein at 4oC, try room temperature instead.
@Adam, I will try your suggestions. I was wondering if proteolysis is possible after elution with 250mM imidazole. Is it because of other protein present in the final elutes?
I am using 50mM sodium phosphate buffer pH 7.5 with 300mM Nacl.
Like what Adam said, you should check the precipitate. Did you dialysis your protein into deionised water or into a buffer with salt? The latter is fine, but protein would be less soluble in pure water.
For quite a few of my proteins, I observe precipitation during purification (usually after leaving in fridge overnight). However, after I remove the precipitate by either filtration or centrifugation, I saw no significant drop in protein concentration (measured by A280). My point is that you should check if you still have enough protein after removing the precipitate. If yes, then just carry on with your purification.
If you are looking to increase the solubility of your protein, addition of arginine is a common technique.
It is very possible that your protein has been precipitated because of dialysis against water, better it would be against your buffer without imidazole. I use a desalting column, as PD10 column, to remove imidazole after NI-NTA chromatography instead of dialysis. it is very useful, it's a fast procedure and it has a good recovery yield. My protein used to precipitate during dialysis processes, but not after PD10 dessalting column. Try it!
Here are few suggestions:
Try to know what is the pI of your protein? you can use the link below to know pI of your protein and some other properties.
http://web.expasy.org/protparam/
You should always keep the pH of buffers atleast 1 unit above or below the pI of your protein.
In general higher pH (more basic) conditions keep proteins from precipitating.
1) Change your buffer to 25 mM Tris and 150 mM NaCl pH 8.0 instead of phosphate buffer for your NiNTA step
2) include 5% glycerol in all your buffers. Glycerol is known to stabilize and help solubility of proteins.
3) Desalting column is a good idea if you have one. If you dont have, you can use concentrators to buffer exchange.
I believe its mostly the selection of buffer, pH and salt that are critical in your case.
Questions for you:
Is your protein a truncated form of a protein or full length, I see from the SDS-PAGE its a very low mol wt protein.
Good Luck.
Venu
If you prepare your protein for a lyophilisation, instead of dialyzing it towards pure water, just dialyze with the buffer you planned for further uses. you will then only need to re-dissolve the protein, which should then work just fine (either in H2O or D2O, for MS or NMR or even neutron diffraction studies)
If you plan on freezing the protein still in water, either flash-freeze without glycerol and keep at -80, or indeed add glycerol and freeze it at -20. Remember however that certain further applications don't appreciate glycerol so much (like, for instance, NMR, and some kinetics studies as it interferes with the correlation time, or the K1, respectively)
In order to resolubilise the precipitated protein, I would advise the use of a stronger denaturing agent (like 8M urea instead of 4, or 4-6M Guanidine HCl) and then dialysis against a standard buffer, but I cannot guarantee your protein will be properly refolded after that (it might be interesting to test, either via CD, fluorescence, activity if it is an enzyme or NMR...)
Are you sure the precipitate (only) contains your protein?
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