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All tags Neuroscience Extraction of amyloid beta from mouse brain

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

DEA extraction isolates non-plaque-associated amyloid beta.

  1. Prepare a solution of 0.2% DEA in 50 mM NaCl.
  2. Homogenize brain using the 0.2% DEA solution at a concentration of 100 mg tissue/mL on ice.
  3. Centrifuge at 100,000 g for 1 h at 4°C (54,000 rpm in 100.3 rotor).
  4. 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.
  5. Neutralized samples can be analyzed by ELISA without further dilution or can be flash-frozen on dry ice and stored at -70°C.
  6. Pellets can be retained and frozen if further extraction is required.
  7. The pellet can also be used for western blot of full-length amyloid precursor protein.

FA extraction

FA extraction isolates deposited plaque-associated amyloid beta.

FA neutralization solution: (1 M Tris base, 0.5 M Na​2HPO4, 0.05% NaN3)

  • 60.57 g Tris base
  • 35.5 g Na​2HPO4 
  • 2.5 mL 10% NaN​3
  • Add H2O to 500 mL; pH is not adjusted; store and use at room temperature
  • CAUTION: Sodium azide (NaN3) is highly toxic
  1. Mix 200 µL 10% (w/v) homogenate into 440 µL cold formic acid (minimum 95%, Sigma, 5-0507) in a microcentrifuge tube.
  2. 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.
  3. Spin 400 µL at 135,000 x g for 1 h at 4°C (50,000 rpm in a TLA 100.3 rotor).
  4. Dilute 210 µL supernatant into 4 mL of room temperature FA neutralization solution. Mix briefly.
  5. 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.
  6. Aliquot and flash freeze on dry ice.
  7. Incubate at 37°C for 5 min prior to loading onto ELISA plates to clarify solution and solubilize precipitate.