Formic Acid Extraction of mouse brains
(Younkin lab - 1999)
1. Weigh frozen brain or hemi-brain. Calculate volume needed to make 150 mg/ml. Add
appropriate volume of 70% Formic Acid to frozen brain and sonicate. We are now
using 5 ml round-bottom tubes for the sonication. Hold the tube in a beaker of ice and
water to keep from overheating. Sonicate until there is no visible structure,
approximately 20 seconds. Then sonicate in 10 sec bursts two or three times. Try to
avoid foaming by keeping sonicator probe in the solution. If it foams, the bubbles will
go down as it sits on ice. Place sonicated brains on ice.
2. Take a uniform volume from each, in order to balance tubes, and spin at 100,000 x g
(32,000 rpm for floor model, 43,000 rpm for table-top) for 1 hour at 4 C. We use the
table-top, so we put 1 ml of each into 1.5 ml thick Eppendorf tubes designed for the
ultra-centrifuge. (You should easily have at least 1-2 ml sonicated solution for each.)
3. Remove supernatant from under lipid layer, leaving small pellet behind. This is the
70% Formic Acid extract that we will assay by ELISA. (We dilute it 1:20 in order to
neutralize it.) We typically aliquot this FA extract into 60 ul aliquots, and freeze at
–80 C.