When my protein was tagged only with the Flag tag , it shows a shift on the western blot. However when I tag it with 6X His and Flag it comes back to the original size. Is this normal?
The Flag tag was used for immunoprecipitation experiments and the HIS tag was added for protein purification from bacterial cells.
SDS-PAGE migrates proteins by their charges. Beside SDS binds to iso-peptide backbones, making protein/peptide negative charged, which allows proteins migrate according to their sizes, proteins migration rate is also influenced by protein shapes and their own charging. Flag tag is negative charge, such negative charge would be neutralised by His tag, making the double tags less different from single tag.
Thanks for the reply . So the Flag tag would migrate slow and give a higher band, while when the His neutralises it shows the original size. Is that correct?
Does it mean that the Hist-tag protein with no flag tag will move faster than expected?
The His tag only protein migrates at the same rate as the endogenous for me.
Yes for me also I have a protein which migrates normal with a hist tag and I have another protein which migrates faster than I expected and this protein is also a hist tag protein.
if u have a claevable site between your 6X his tag & protein, after purification you can cleave and check the migration of band with or without his tag.
plus the exp of charges, which makes flag+His-protein run better than Flag-protein is good and i second that too