I have purified a EGFP-His tagged protein which was there in pET15b vector using Ni-NTA column. The concentration obtained was way too high around 5mg/ml. Before the running the SDS-PAGE the sample was boiled for 10 min. But, on SDS-PAGE I have obtained the the molecular weight of 60 kDa instead of 30 kDa which is the molecular weight of EGFP.
Native GFP can be dimeric in such a high concentrated solutions ( 5 mg/mL), concentrate it only upto or below 2 mg/ml and see if this helps. For current prep, use sodium dithionite and check if you see band at the position which you expect to see
Dear Puneeta,
I confirm Mahesh answer. The expressed EGFP is a dimeric protein.
I suggest you performing reduced SDS-PAGE.
You can see the attached references about GFP oligomerization.
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