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Denaturing IB in 8 M Urea - (Oct/14/2010 )

Dear all,
I am purifying a protein of mol.wt of 22KDa in E.coli BL21(DE3)pLys. as my protein is overexpressed as inclusion bodies, i have to denature it in 8M urea solution. but once i keep it for stirring in cold room i m getting precipitates after some time.. wat r those precipitates.. is it urea ? or will it be both protein and urea..? if i load the solution for dialysis should i load the suprnatant only leaving off the precipitate or whole solution by dissoving the precipitates..? plz suggest a way.. this is my procedure..

Make 8 M urea solution. Total volume of solution will be however many IBs you have
(mg) so much mL (eg 530 mg IB -530 mL 8 M solution)
Heat urea solution
Dissolve in only half the volume of water b/c urea takes up volume.
add 14.2 mL BME/1L urea, this comes out to be 0.18 M BME.
Dissolve inclusion bodies on ice with stirring.
followed by dialysis.

Thanks in advance.

-mmmm-

don't heat urea. it will decompose (even at slightly higher than room temperature). decomposed urea can carbamylate your protein.

if the precipitate looks like crystals then it is urea. urea will crystallize at cold room temperatures.

-mdfenko-

mdfenko on Fri Oct 15 14:09:22 2010 said:


don't heat urea. it will decompose (even at slightly higher than room temperature). decomposed urea can carbamylate your protein.

if the precipitate looks like crystals then it is urea. urea will crystallize at cold room temperatures.


Thank you very much..i will try with out heating next time.. as u said the precipitate looks like crystal. so i can leave off that and load the supernatant in to dialysis bag.. am i right ?

-mmmm-

mmmm on Sat Oct 16 08:15:30 2010 said:


Thank you very much..i will try with out heating next time.. as u said the precipitate looks like crystal. so i can leave off that and load the supernatant in to dialysis bag.. am i right ?

i would allow the crystals to dissolve at room temperature before dialysis to ensure that no more than normal is lost during processing.

-mdfenko-