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sample precipitation problem in western blotting


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#1 yobou

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Posted 24 March 2012 - 03:39 AM

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

#2 proteaMatt

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Posted 27 March 2012 - 06:21 AM

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.
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#3 mdfenko

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Posted 27 March 2012 - 07:53 AM

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