Posted 26 August 2010 - 09:03 PM
Im trying to purify alkaline phosphatase enzyme that has been overexpressed in E. coli BL21DE3. The protein formed inclusion bodies upon IPTG induction. I was able to isolate and purify the protein in the inclusion bodies using denaturing Nickel affinity chromatography. Then i used dialysis to remove the denaturant (8 M urea)
1. After i dialysed the unfolded protein for 48 hrs in a cold room. next day i was able to see a white precipitate at the bottom of the dialysis bag. I assumed it is the salt, but my colleague told it was the refolded protein itself.
2. I performed the assay after concentrating the dialysed samples using a speed vac. Still that white precipitate was visible.However the assay worked.
Was that white precipitate a salt or refolded protein?. Is dialysis not enough to remove urea?
Posted 26 August 2010 - 10:41 PM
talent does what it can
genius does what it must
i used to do what i got paid to do