I have been purifying proteins which have an N-terminal His-tag using Ni-NTA affinity chromatography. I carried out size exclusion chromatography using Superdex HiLoad, Superdex, and Superose columns. I also carried out dialysis of the Ni-NTA purified proteins.
Both the above experiments were performed for buffer exchange and to remove non-specific proteins.
When I ran SDS-PAGE gels for the Ni-NTA purified and dialysed proteins, I could see a single band corresponding to the protein of my interest. Multiple bands beneath the protein of interest could be seen on SDS-PAGE gel for the SEC fractions.
I used appropriate controls to rule out the possibility of degradation due to prolonged exposure at room temperature and the effect of varying salt concentrations.
Please help... to all the protein purification experts out there!!
There can be many reasons like sample heterogeneity, aggregation, degradation of sample, cross-contamination with other protein sample
Sonia Sharma I have cross-checked the reading frames in all of the constructs and there happens to be no mispriming. As for aggregation, the single peak on chromatogram comes within the resolving range. Also, if there is an aggregation, is there any way to confirm it?
If there was cross-contamination, it would appear even after Ni-NTA purification as well?
I have considered degradation to be a possibility, but I can't understand why is it only observed after SEC.
Aggregates often form more readily at higher concentrations. Perform SEC experiments at different sample concentrations to see if the number or intensity of bands changes.
If you have an antibody (preferably polyclonal) against your target do a western to check for proteolysis.
Engelbert Buxbaum The proteins have an N-term His tag, I have performed Western Blot using anti-His antibody which gives a band corresponding to the protein of interest. None of the bands underneath are seen.
What is the SEC phase composition and details of your application? Do you see these Lower MW proteins even after using different SEC resins/columns? Is that reproducible?
Possibly the degradation products or contamination is the case either coming out through the Ni-NTA during the first step of purification or they are being formed during SEC application. Assuming reagent blank injection for the SEC columns gives a straight baseline (just noise) to exclude the column contamination that may remain from the previous application.
If the proteolytic cleavage did occur near the N-terminus, you could not see the fragments with anti-His antibodies. Thats also why polyclonal ab are better than monoclonal for this application.
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