The pure bioactive protein/peptide isolated from marine tissue in ion exchange column is showing two peaks in HPLC but a single band in SDS PAGE. How i can take these results? Whether the isolated protein is pure or not?
HPLC can resolve minor alteration in peptide like the oxidation, deamidation, disulfide reduction, while SDS PAGE can't. So there is a possiblity that it can happen with a pure protein. I have not run RP-HPLC, but have learnt this from a manual which says so. You can find it here.
http://www.hplc.eu/Downloads/ACE_Guide_Peptides.pdf
MALDI TOF analysis of your HPLC purified sample can assist in getting an inference on the above stated alterations.
If your HPLC system is run properly, the answer is simple: Your protein consists of at least two different compounds. However, in the field of proteins, it is not trivial to define, what "pure" may mean. Some proteins consist of isoforms, others can oxidize easily, as Mangal already mentioned. Most "pure" proteins (even pharmaceuticals) consist of several similar compounds, which are similar, but not identical in a chemical sense. Since ion exchange and SDS-PAGE are separation techniques of relatively low resolution, it is not surprising that you get more peaks in RP chromatography.
If you've found two peaks in ion-exchange chromatography and single peak in SDS-PAGE it could mean that your protein has at least two isoforms (different pI value). You can confirm it by IEF run.
- Badges
- Science method