INCLUSION BODY SOLUBILISATION PROBLEM

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chitra
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INCLUSION BODY SOLUBILISATION PROBLEM

 hello all,

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?

Chitra