protein aggegation or? - protein in the wells of native gel (Jun/23/2004 )
We observed unusual behaviour of our insect cell purified proteins (members of NFkappaB) - they do not enter 5% AA native gels and once bound to probe barely enter EMSA gel since radioactivity stays in the wells. (pIE is for one 6.5, other 7.8). To the contrary, the same proteins, when produced in E.Coli, enter native gels and do nice shifts in EMSA!
We have played with various protein amounts, added detergents ( increasing concentrations of NP40, sarkosyl or SDS), NaCl (150mM), MgCl... Nothing worked except when 0.1% SDS was added which seemed to push p65 slightly in the gel, but too high for the expected size, when compared to native protein marker. Strangely, conditions for purification from E.Coli and insect cells are identical and dyalized/stored in the same buffer...
Is anybody having any sparking idea?
The insect cells have heavily modified your protein-probably through extensive glycosylations.
Hmm, NF-kB was not shown to be glycosylated, neither goes through the secretion pathway so I don't think it could be the reason?
If your insect cell purified NF-kB is not modified according to post-purification LC-MS, ES-MS or GC, then it may be a different reason that the protein is not entering the gel.
Please donot discount the possibility of modification though; because NF-kB will be inherently toxic to the insect cell line due to its dynamic biological capabilities and the only way a cell expressing a foreign gene can neutralize the toxicity is by modifying the expressed agent.
Are you sure it is not modified? I am not speaking about the native protein or known research data about NF-kB; just curious about the protein coming off the insect cell line.