lysis is not translucent after urea treatment - need help with His tag fusion protein purification (Dec/03/2005 )

HI,

I am tring to purify insoluble his tagged protein according to Qiagen Ni-NTA argrose handbook. I harvested cells from 20ml culture and resuspend in Buffer B (8M urea, 10mM Tris, 100mM NaH2PO4 PH. 8.0), vortex for about 1 hour in room temperature, but the cell culture was still cloudy. What happened?

Hi,
Try preparing frest Urea buffer. If it is old, the urea breaks down and can cause some nasty unwanted ionic interactions with your protein.

Also, make sure you freeze-thaw a few times (or overnight) to weaken the cell walls before lysis.

Good luck!

Thank you.

I waited o/n and first sonicated then use urea to solubalize, and it worked.