- Mix 200 μl 10% (w/v) homogenate into 440 μl cold formic acid (minimum 95%, Sigma, F-0507) in a microcentrifuge tube.
- 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.
- Spin 400 μl at 135,000 x g for 1 hr at 4°C (50,000 rpm in a TLA 100.3 rotor).
- Dilute 210 μl supernatant into 4 ml of room temp 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.
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.
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