I subject NiNTA purified proteins to size exclusion chromatography to get rid of any impurities and identify monomers. I'm not entirely sure what the best approach is for further downstream analyses to make sure I've indeed purified my protein of interest. I've considered ELISA and western blot but there are a multitude of primary antibodies that recognize this protein and I'm unsure of which might be best.
To find out if protein of interest is highly pure, you got to do SDS-PAGE followed by silver staining. However, IMAC (especially Ni++ resin; Co++ is better) seldom yields greater than 90-95% pure protein.
The Best way to find the purity of elution, as Seyed A Ali said, is SDS-PAGE. ELISA is not recommended for checking purification. However, western blot is good because before cross immunization, you must run a PAGE and this can be helpful.
Tryptic digest and MS will conclusively identify your protein band from SDS-PAGE but intact MS will ID your protein and tell you if you have any degradation by proteolysis etc. If your protein will 'fly' intact then go for that, otherwise digest and MS/ID the fragments.
Thank you for the suggestions. Evidently SDS-PAGE is the best option. My FPLC profile had several peaks, which I wasn't expecting. And for one particular profile I saw a very broad peak and I wasn't sure which fractions to collect.
Of course SDS-PAGE is a very good start-you can see the relative purity and approximate size but it would not stop you purifying, for example, an endogenous host protein ( from E.coli, 293 cell etc...) th esame size as your target protein. If you are working with a protein you are familiar with, or one that is very well expressed and can easily be discerned from a host protein, then that may be fine but if you are purifying a new protein I would still recommend getting a positive ID of your protein by MS as early as possible.
Also for the first prep of a protein it is worth running an SDS-PAGE gel of the IMAC fractions before continuing with additional chromatographic steps, but do try to do this as quickly as possible as some proteins really do not like to sit in high concentrations of imidazole. If your protein looks really dirty after IMAC then SEC may not help so much, think of modifying your IMAC and playing with increasing imidazole concentrations in your wash steps or a gradient elution can help in some cases.
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