As title mentioned, I have done separation by centrifugation right after the mechanical lysis since I knew the protein of interest was chiefly appeared as inclusion bodies. This was my first time to use denature-renature method to obtain the product. After letting the stir bar spin inside to break up the pellet for 4 days (I had holidays last week) in Tris buffer with 8 M urea, it became an opaque and white solution. A colleague said that I could try lowering the pH.
What could be the reason behind the outcome? Simply because of pH?
Formulation: Tris 20 mM, pH 8@20°C / NaCl 0.5 M / Urea 8 M
4 days is unnecessary that also with 8M urea- people do few hours to overnight. First you search the half time of denaturation of this protein. You can simply repeat this stuff with ph 7.5 and less denaturation time. Lowering pH below in acidic range may be or may not be useful - what is pi of your protein ?
Did you wash the inclusion bodies before adding 8 M urea? How much volume of urea solution are you using with respect to the original suspension volume?
I agree with Omar's question, almost repeated it before I saw his entry.
Also, is it a very charged protein, lots of basic or acidic residues? You may perhaps increase the buffer capacity a bit to 30 to 50mM Tris.
When you left the solution stirring over 4 days, was there a tight lid on the container? If there was too much evaporation of water, the whitish stuff could be urea crystals.
Sorry for the late reply.
Omar Gonzalez-Ortega No; 60 mL of urea solution
Achim Recktenwald they constitute 21%; in Falcon tubes, screw closure, therefore no change of volume
The whitish stuff could also be lipids - the washing step for the IB is important.
Hi Mr Achim Recktenwald ,
I found a protocol (Curr Protoc Protein Sci. 2004 Nov;Chapter 6:Unit 6.3.) mentioning that the pellet was washed with urea and Triton X-100 so as to remove cell membrane materials. Will Triton X-100 surround misfolded proteins as well?
Yes, this can be a problem; depends on the protein. But the 8M urea should remove most of it.
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