Directly conjugated antibodies for immunofluorescence

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DEA Extraction of [soluble] Amyloid beta from Mouse Brain

Extraction of non-plaque-associated [soluble] amyloid beta from brain homogenates

  1. Prepare a solution of 0.2% DEA in 50 mM NaCl.
  2. 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.
  3. Centrifuge at 100,000 g for 1 hr at 4°C.  (54,000 rpm in 100.3 rotor).
  4. Remove supernatant and neutralize by adding 1/10th volume 0.5 M Tris HCl pH 6.8. Vortex gently.
  5. Neutralized samples can be analyzed for amyloid beta by ELISA without further dilution.
  6. Samples can be flash-frozen on dry ice.
  7. Pellets can be retained and frozen if further extraction is required.
  8. 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