sample precipitation problem in western blotting
Posted 24 March 2012 - 03:39 AM
I am facing a strange problem in western blotting. After I start electrophoresis of samples, I find a precipitate in the bottom of some wells that does not enter the gel easily. I have to reduce the voltage dramatically until it disappears and enter the stacking gel but this takes about an hour. I am using RIPA buffer for sample lysis (50mM TRIS, pH8, 150mM NaCl, 1% Igepal, 0.1% SDS, and 0.5% deoxychlate)+commercially available protease and phophatase inhibotors. I boil samples at 98 degrees for 3 minutes in 5X SDS loading dye containing beta mercaptoethanol.
what could be the reason for this?
thanks a lot
Posted 27 March 2012 - 06:21 AM
Posted 27 March 2012 - 07:53 AM
genius does what it must
i do what i get paid to do