- Prepare a solution of 0.2% DEA in 50 mM NaCl.
- Homogenize brain in 0.2% DEA solution at a concentration of 100 mg tissue/ml on ice. For example, 100 μl 0.2% DEA/10 mg brain, homogenized on ice with small blue homogenizer.
- Centrifuge at 100,000 g for 1 hr at 4°C. (54,000 rpm in 100.3 rotor).
- Remove supernatant and neutralize by adding 1/10th volume 0.5 M Tris HCl pH 6.8. Vortex gently.
- Neutralized samples can be analyzed for amyloid beta by ELISA without further dilution.
- Samples can be flash-frozen on dry ice.
- Pellets can be retained and frozen if further extraction is required.
- The pellet can also be used for WB of FL-APP.
Dilution guidance notes:
For amyloid beta [c] in brain -
if value = 20 pM
20 pmole/Liter = 20 pmole/1000g
x 12 for dilution = 240 pmole/1000g = 0.24 pmol/g
Dilution is 12 because we take 100 mg of brain in 1 mL 0.2% DEA solution plus 100 μl Tris
100 mg in 1.1 ml = 1:12 dilution