Hi Megha,

You could  make your buffer solutions at room temperature, so that the 8M urea dissolves completely. During solubilization of the protein, keep the tube on a shaker or agitate the solution using a magnetic strirrer. This will prevent any crystallization of the urea. In any case, urea crystallizes very slowly, even at 4 degrees C, as already mentioned .

Another issue is the solubilization time. You've mentioned that you do it only for 1 hour. In the  proteins I have worked with, I have had to solubilize overnight to get a reasonable yield. Of course this could be different for each protein.

Hope this helps

Ganesh

On Wed, 2 Dec 2009 20:16:09 -0800, megha goyal <[log in to unmask]> wrote:

Hi all,

 

We use 8M urea solubilization buffer for our protein in inclusion bodies and recommended temperature is 10-15º C. but in 8M conc the urea does not dissolve and is in crystalline form only, will it have any effect on solubilzation efficiency. Our solubilization time is 1 Hr and after that we centrifuge and use the supernatant for refolding via dialysis. however the pellet after centrifugation of solubilzation show presence of our protein on sds page analysis. what should we do so that the process of solubilization is complete and our protein is not lost in pellet.

 

thanks in anticipation.

 

meg