Tumor protein precipitation at denaturing step? - (Sep/21/2012 )
I processed some tumor samples to be used for a Western Blot. I homogenized the tumors in lysis buffer with a stirring bead, prepared 50 ug/well for 10 repeats. I made a total volume of 200 including 40 uL 4x loading dye and the remainder DI water. I mixed the samples in a microfuge tube and put them on a 100 C heating block for 5 minutes. When I took the tubes off of the heating block though, there was a dark blue precipitate on the bottom of the tube and a clear pink supernatant.
A couple things:
I had a total of 300 ug of protein/tube
A couple of the tumors were rather bloody
I was told not to use a reducing agent
I ran the samples anyways to see what I would get and I got irregular banding when I stained with Ponceau...no banding seen upon imaging as well
Any thoughts as to what is going on to produce this precipitate? Or what I can do to prevent it in the future?
A few things - what is in your lysis buffer? What was the pH of the lysis buffer and the loading dye? what are the components of the loading dye?