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Extraction of amyloid beta from mouse brain
Discover the two procedures, soluble (DEA) and insoluble (FA), for the extraction of amyloid beta from mouse brain.
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This protocol is for the extraction of two fractions; soluble (extracted using diethylamine, DEA) and insoluble (extracted with formic acid, FA).
DEA extraction isolates non-plaque-associated amyloid beta.
- Prepare a solution of 0.2% DEA in 50 mM NaCl.
- Homogenize brain using the 0.2% DEA solution at a concentration of 100 mg tissue/mL on ice.
- Centrifuge at 100,000 g for 1 h at 4°C (54,000 rpm in 100.3 rotor).
- Take the supernatant, which contains the soluble fraction, and neutralize by adding 1/10 volume 0.5 M Tris HCl pH 6.8. Vortex gently.
- Neutralized samples can be analyzed by ELISA without further dilution or can be flash-frozen on dry ice and stored at -70°C.
- Pellets can be retained and frozen if further extraction is required.
- The pellet can also be used for western blot of full-length amyloid precursor protein.
FA extraction isolates deposited plaque-associated amyloid beta.
FA neutralization solution: (1 M Tris base, 0.5 M Na2HPO4, 0.05% NaN3)
- 60.57 g Tris base
- 35.5 g Na2HPO4
- 2.5 mL 10% NaN3
- Add H2O to 500 mL; pH is not adjusted; store and use at room temperature
- CAUTION: Sodium azide (NaN3) is highly toxic
- Mix 200 µL 10% (w/v) homogenate into 440 µL cold formic acid (minimum 95%, Sigma, 5-0507) in a microcentrifuge tube.
- Sonicate each sample individually for 1 min on ice. Immerse the tip of the probe in the sample, then move tube up and down while sonicating.
- Spin 400 µL at 135,000 x g for 1 h at 4°C (50,000 rpm in a TLA 100.3 rotor).
- Dilute 210 µL supernatant into 4 mL of room temperature FA neutralization solution. Mix briefly.
- FA neutralization solution is stored and used at room temperature, as a precipitate will form if it is stored at 4°C or placed on ice.
- Aliquot and flash freeze on dry ice.
- Incubate at 37°C for 5 min prior to loading onto ELISA plates to clarify solution and solubilize precipitate.