I have theoretical PI of my protein from sequence data which is 5.59. I have tried anion exchanger (DEAE-macrosep) starting with buffer at pH 8 and then reducing it down to 6 followed by NaCl elution (250mM-1M). However could not find any activity in the final elution. I further tried a cation exchanger (Macrosep high sulfonated) column starting with pH 4.5 and increasing it upto pH 6.5 followed by elution with 500mM and 1M NaCl. In this case also, I could not find any protein activity in the elutions. I pooled and concentrated the final elution samples while carrying out assay. I am unable to figure out. Before ion-exchange I had partially purified the protein using Ammonium sulphate precipitation. Kindly suggest an alternative. I am not sure of my actual protein size, but it shows activity in retentate when passed through 100KD membrane.
Please clarify whether there was activity in the sample that was applied to the column, and if so, whether the active protein bound to the column so that there was no activity in the flow-through fractions.
If you started with an ammonium sulfate precipitate, did you desalt it or dialyze it to remove the residual ammonium sulfate in the resuspended pellet prior to applying it to the column? If not, the high salt may have prevented the protein from binding to the column.
Some of the pH conditions you used, such as 4.5, are pretty far from neutral and could have damaged the protein. Try to do the chromatography at neutral or near-neutral pH, eluting with a salt gradient.
Is there any reason to think that your protein functions as part of a complex with another protein or a small molecule? If so, these components may have been separated into different fractions by the chromatography, causing a loss of activity.
Did you do the chromatography at 4oC to prevent denaturation or proteolysis?
Why not to try ion exchange chromatography at constant pH first? Works much better for proteins which experimental pI is unknown. Just bind your protein at a low salt level (let's say 10-20 mM) and gradually increase NaCl up to 1M concentration upon elution. You can use your starting buffers (pH 8 and 4.5).
I can try that. But, in the earlier case as well had the protein been bound to the column, it should have eluted at such high NaCl concentrations.
Anna Belorusova might be correct, you better try ion exchange chromatography at pH 7.4 . Once if you find your protein in eluate, collect all the fractions and concentrate it and remove the NaCl by millipore dialysis ( buffer exchange ) with appropriate buffer. You might be knew that Ionic strength of protein solution may effects the protein activity.
The best technical support will be from GE - since they sell all kinds of related products. (1-800-526-3593). We run Ion exchange column by running a gradient between 2 buffers - the second buffer of higher salt concentration, both same pH.
Please clarify whether there was activity in the sample that was applied to the column, and if so, whether the active protein bound to the column so that there was no activity in the flow-through fractions.
If you started with an ammonium sulfate precipitate, did you desalt it or dialyze it to remove the residual ammonium sulfate in the resuspended pellet prior to applying it to the column? If not, the high salt may have prevented the protein from binding to the column.
Some of the pH conditions you used, such as 4.5, are pretty far from neutral and could have damaged the protein. Try to do the chromatography at neutral or near-neutral pH, eluting with a salt gradient.
Is there any reason to think that your protein functions as part of a complex with another protein or a small molecule? If so, these components may have been separated into different fractions by the chromatography, causing a loss of activity.
Did you do the chromatography at 4oC to prevent denaturation or proteolysis?
I had desalted before applying the sample into the column. Besides, there was activity in the sample before column chromatography step. My protein of interest does not require any other protein to function. It only requires a cofactor for enzyme assay. I will carry out the elution as suggested by you guys at neutral pH and let you know the results.
The sample applied needs clarification on whether there is activity before application, and the idea of using starting buffer of pH 7-7.4 is a good one, however, ensure that your gradient elution is properly taken care of. is your protein bound or unbound after the intial step? or you try Acetone precipitation and dialysis method instead of ammonium sulphate and dialysis. Ensure you work at lower temperature as suggested by Adam B Shapiro
@Frank Abimbola Ogundolie : There was activity before applying the protein sample onto column. In the flowthrough I obtained very less activity compared to initial one after concentrating the pooled samples. I can not exactly quantify whether the protein was completely bound or unbound. But, from activity in flow through it looks like some protein did bind to column.
Try eluting with a rapid salt step (something like 1M NaCl), to reduce dilution and get a more concentrated eluate, check for activity and then work on optimizing the elution gradient.
I agree with Prof.Adam raised points needs some clarifications
Not finding activity in the final elution does not mean your protein is not there, rigth (SDS-PAGE).
I just figure out:
1. Could you consider High salt content is affecting the way you evaluate protein activity in your final elutions?Are you desalting your elutions if not,Try desalting your sample, maybe use a protein refolding solution (in case your protein is denatured).
2. Have you consider in create monoclonal o polyclonal (better , I consider in your case) antibodies against synthetic peptides derived from your protein sequences (try to consider linear epitopes if is posible)?, so in the future you can assert the localization of your protein in your protein elution even if it is partially degraded (SDS-PAGE, Immunoblotting) or denatured, that should works.
3. 4 Celcius , Proteases inhibitor, for sure most be in your in your initial tests.
Best, I hope this could help you.
Membrane-based separators such as DEAE-macrosep can bind unspecifically. Proteins re-suspended after ammonium sulfate often get aggregated. I will recommend to try good old DEAE cellulose column instead and run under conditions you tried already with macrosep.
Maybe the problem has been solved already, but you could try Free-Flow electrophoresis. There you don't have a matrix that can interact with your protein and the whole system is cooled. This should give you the mildest conditions possible for the separation.
@Christopher Timm: Thanks for your reply. The purification has already been completed
First you should desalt your sample after ammonium sulfate ppt.by either dialysis or gel flirtation on sephadex G25 before you used DEAE step.Second you could try gel flirtation to separate your protein according to size using for example sephadex G75 or G100 depend upon MW of your protein. You also may try isoelectricfocusing to separate your protein according to PI since you know it. Wishing you the best
- Badges
- Science method