FA Extraction of [insoluble] Amyloid beta from Mouse Brain

FA (formic acid) extraction of deposited plaque-associated [insoluble] amyloid beta from brain homogenates

  1. Mix 200 μl 10% (w/v) homogenate into 440 μl cold formic acid (minimum 95%, Sigma, F-0507) in a microcentrifuge tube.
  2. Sonicate each sample individually for one minute 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 hr at 4°C (50,000 rpm in a TLA 100.3 rotor).
  4. Dilute 210 μl supernatant into 4 ml of room temp 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.

FA Neutralization Solution

(1M Tris base, 0.5 M Na2HPO4, 0.05% NaN3)

  • 60.57 g Tris base
  • 35.50 g Na2HPO4
  • 2.5 ml  10% NaN3
  • H2O to 500 ml; pH is not adjusted; store and use at room temperature.
  • CAUTION:  Sodium azide (NaN3) is highly toxic.

Notes:

Neutralized material is usually diluted at least 1:2 with buffer EC prior to loading onto ELISA plates, as a precipitate forms when samples are loaded neat. If analyzing tissue with a lot of plaques, you need larger dilution factors to analyze amyloid beta levels within the range of the standard curve. To determine amyloid beta [c] in samples, multiply interpolated values of material loaded on plate by 64.15