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sample precipitation problem in western blotting - (Mar/24/2012 )

dear all
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


You might try reducing the protein concentration of your sample or reduce boiling temperature and time. I have found that sometimes if I heat the sample too long I end up with protein aggregates (most often albumin) that are very difficult to get to enter the gel.


if you reduce boiling (incubation) temperature then you may need to increase time. incubation at 70C should be for 10-20 minutes.