I purified my lectin from a seed by ion exchange chromatography followed by gel filtration chromatography.The eluted fractions after gel filtration chromatography were used to perform a non-reducing SDS-PAGE. I obtained 2 bands for my pure protein.After which, I performed a NATIVE PAGE (Resolving gel-10% and Stacking gel - 4%).However I see a faint streaky pattern in the gel and there seems to be a slight aggregation in the well itself. No bands in the gel.Could it be that my protein is huge and I need to use a lower resolving gel %. Could anyone give me some suggestions cos its a mystery as to why I cant see any bands in the NATIVE PAGE. I used BSA as a standard for which I have obtained a good prominent band.
Dear Deepti Madayi,
There are several reasons that may explain the running behavior that you observe in Native Page (assuming that the protein you obtained is pure). One, as you mention can be an aggregation effect. But it is not the only reason. Many proteins are not monomers under native conditions, but they rather oligomerize, forming big complexes that do not enter the well on native gels. In some other cases, the amino acid composition, or posttranslational modifications affect the running pattern. Normally, highly acidic proteins are unstructured under native conditions and they run at a higher molecular weight than the predicted size (you can observe that even under denaturing conditions). I am not familiar with lectin, then I do not know if any of those explanations fit with your case. In any case I would try to reduce the percentage of your gel. In my experience, 5 % gel works well to resolve the proteins I work with.
Best
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