I have problems with my experiment, I am trying to extract whole protein from mammalian cells and the target protein can be located in 3 forms, the unsoluable (aggregation, inclusion body,P1),oligomers (soluable but larger, P2), and the monomer.
I used RIPA buffer to lysis the cells and centrifuge 1000g 10min to collect the pellet (containing unsoluable aggregation), the supernantant (containing monomers and oligomers applied to detect protein expression level) was centrifuged at 16,000g 1 hour to harvest pellet (oligomers).
In the end. I did the W.B., the P1 and the P2 had the similar bands, with sample 1 and 2, which 1 should have no bands in P1 because it will not form any aggregation.
Any suggestion will help, wish for your help.