I have a protein that forms inclusion bodies, so i did some small scale tesing on urea and it seem to solubilise with 4M Urea. I upscaled and the protein purified nicely, unfortunately i lost the protein after gf due to some silly mistakes. Ever since I have been trying to repeat with no luck.
The big problem i am finding is the solubilisation of my inclusion body stage with UREA. I resuspend the pellet with the urea and buffer but the protein stays in the pellet. This is even after leaving it overnight at room temperature. I runa gell and i can lots of protein in the pellet and not the supernatant. WHAT COULD I BE DOING WRONG? Is this an expression problem maybe?