I am purifying my L-Asparaginase (with 6 his-tag) using Ni-nta chromatography, and imidazole 500 mM + potassium phosphate 0.1 M as and elution buffer and pH of 7.5, and then I check the activity using nessler's reaction with asparagine as a substrate to determine which elution fraction has my active protein.
I put it in a dialysis tubing and leave in potassium phosphate buffer pH of 7.5 (it volume is 200 times the volume of fraction with activity) and leave it at 4°C overnight.
Next day in the morning, no activity is observed.
N.B. Dialysis tubing is the only available dialysis method.
The protein is stable at room temperature (if this information is needed).
I need to remove the imidazole because it interferes with cell viability applications.
Many thanks and regards.
Your protein certainly isn’t vanishing. it is either sticking to the dialysis membrane or, more likely, has simply been inactivated by the dialysis conditions. I don’t know anything about this enzyme but some general points to consider would be the type of buffer used, pH and ionic strength. I’d probably start by looking at the conditions used for assaying the enzyme. What buffer and salts are used for that purpose? They might be what you should have in your column buffers and dialysis buffers. Next, you might consider what could be missing. Glycerol can sometimes be helpful. Finally, if the concentration of the protein is very low the enzyme could simply be lost on surfaces. You could try adding BSA to the enzyme prior to dialysis. This might enhance stability too.
It is likely that the proteins were not properly bound to the membrane of the dialysis tubing or that the dialysis tubing was not properly sealed. If the proteins were not tightly bound to the membrane, they could be washed away by the buffer, resulting in the loss of the proteins. Additionally, if the dialysis tubing was not properly sealed, the proteins could leak out through the tubing and be lost.
Dear Mark,
I think your protein is denatured by the imidazole. This is normal in the case of L-Asparaginase. Renature the protein in its optimum buffer with a lower molarity of the buffer (20 or 50 mM). After dialysis, recover the protein in the reducing buffer so that the enzyme is not diluted. Do desalting of protein.
Good luck
Did you check on SDS-PAGE? That should tell you if the protein is there or not. If the imidazole exposure is a problem you might want to look at other options for elution. First of all you could use lower imidazole concentration - most proteins will be eluted with less than 500 mM. Try elution with a gradient. It may be advantageous to do buffer exchange on a column instead if imidazole exposure time is important.
Another possibility is elution by lowering pH. You can also try elution by ammonium chloride (gradient up to 2 M).
Hi, I agree with Bo's comments above.
Another alternative to maybe use less imidazole for elution is to use a Co-charged resin (e.g. WorkBeads 40 Co-NTA from Bio-Works) as Co-charged resins usually bind His-tagged proteins not as strong as Ni-charged resins (and also often gives you a more pure eluted protein).
Also to do the removal as fast as possible I also suggest to do a quick desalting on a column use e.g. GoBio Mini Desalt 1 mL column (Bio-Works) to do a quick test and then you can easily scale up to larger prepacked Desalt columns (I am not sure in what scale you are working).
Do you know the molecular weight of the L-Asparaginase that is eluted in the Imidazole, as I read the Molecular weight can vary by species from 80,000 down to 27,000 daltons? The dialysis membrane you use is it rated to retain a molecular weight of the L-Asparaginase? Have you considered concentrationing your L-Asparaginase and then running a simple Size exclusion Chromatography to remove the Imidazole (de-salting)? Is the pH of the elution close to the pI of the L-Asparaginase?
Dear Mark,
I hope you are in a great health. Here are some tips could help to solve this issue.
1. First you should know exactly the molecular weight of L-Asparaginase to help you in selecting a proper dialysis bag or tube which will have pores smaller than size of your protein.
2. Try to perform your purification using different conc. of imidazole less than 500 mM (100,200,300,400) within elution buffer then check the activity for your enzyme per diff conc.
3. Dialysis at room temperature will be helpful as your protein is stable in this condition.
1. You might need doing SDS-PAGE for checking whether your protein exist, or detecting your protein by absorption at 280 nm. So far, your result just showed that the activity of your protein is lost, and existence of protein is unknown.
The lost of protein's activity might be caused by the high concentration of imidazole in buffer.
2. Nessler' reaction need KOH, which may be offset by KxHyPO4, or the KOH might lead to the denaturation of your protein. Ref: https://protonstalk.com/chemistry/nessler-reagent/
Check the molecular weight cut-off of your dialysis tubing vs the molecular weight of your protein. MWCO tubing should be
Thank you all very much, I tried elution with dropping pH, it worked a little bit, but I needed the protein more concentrated than that (this method eluted many fractions containg the protein, rather than only one or two with high concentration). After practically taking all your answers in consideration, apparently the problem was the very high conc of imidazole, and the improper sealing of the dialysis bag.
Thanks for the update Mark.
In future it could be worth 'scouting' different elution conditions by testing out binding and eluting efficiencies at small scale (even 10 - 20 µL resin in an eppendorf tube, bind and elute in batch mode to find optimum buffer conditions (so long as the protein is safe!)).
Sealing the bags, I used to use sandwich bag clips like these: https://www.ikea.com/gb/en/p/bevara-sealing-clip-set-of-30-mixed-colours-mixed-sizes-10339171/
and always fill the tubing first with water to rinse out wetting agent, then with the buffer you are using, can check for leaks / seal.
Best of luck,
P
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