Removing fat from tissue preps

[Paul S. Brookes.]

One thing you might consider is the speed of the spin.  Microcentrifuges 
are not much good for preparing anything for SDS PAGE.   You can only pull 
about 10-15,000 x g in a microcentrifuge at 14,000 rpm top speed.  If you 
use an ultracentrifuge you should be able to pull at least 60,000 x g, 
which will make the fat layer much more pronounced and easier to pipet away.

Also if concentration is not a problem, try homogenising in a greater 
volume of buffer to improve the separation.  100mg of tissue in 1ml buffer 
is going to give you such a high protein concentration that the fat might 
be sticking to your proteins, hence its inability to be removed.  Is there 
detergent in your buffer?  This might also aid in removing, or at least 
emulsifying, the fat.

Regards
PSB

_________________________________________
Dr. Paul S. Brookes.            (brookes at uab.edu)
UAB Department of Pathology,   G004 Volker Hall
1670 University Blvd., Birmingham AL 35294 USA
Tel (001) 205 934 1915     Fax (001) 205 934 1775
http://peir.path.uab.edu/brookes

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