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protein refolding

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#1 chn09



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Posted 26 August 2010 - 09:03 PM

Hello everyone.
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?
pls help

#2 mdfenko


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Posted 26 August 2010 - 10:41 PM

it was most likely your protein, but not all of it (that's why your assay worked). you may avoid this precipitation if you reduce urea stepwise and slowly.

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