Following are procedures which researchers have followed for handling beta amyloid in tissue and cell samples:
Kienlen-Campard, P, S. Miolet, B. Tasiaux, and J.-N. Octave (2002) Intracellular amyloid beta 1-42, but not extracellular soluble amyloid beta peptides, induces neuronal apoptosis. J. Biol. Chem. 277(18):15666-15670. These authors performed formic acid extraction of whole cells. Neurons (approximately 107) were scraped in ice cold PBS. Cell pellets were solubilized in 300 microliters of 70% formic acid. Formic acid-solubilized cell pellets were cleared by centrifuging at 16,000 x g for 5 minutes at 4 degrees C, and the supernatants were centrifuged at 21,000 x g for 20 minutes at 4 degrees C. The supernatants were vacuum dried and the resulting pellet was resuspended in 1 mL of alkaline carbonate buffer (2% Na2CO3, 0.1 N NaOH) and centrifuged 16,000 x g for 3 minutes at 4 degrees C. Protein concentration was determined by the BCA assay. For immunoprecipitation, 800 microliters of the supernatant was neutralized with 800 microliters of 1 M Tris-HCl, pH 6.8, and diluted 1:3 in H2O containing 10% FCS, 0.5% Triton X-100, and 0.5% Nonident P-40 (final concentrations). Beta amyloid 1-40 and Beta amyloid 1-42 concentrations were determined by ELISA using 100 microliters of neutralized extract.
Fagan, A.M., M. Watson, M. Parsadanian, K.R. Bales, S.M. Paul, and D.M. Holtzman (2002) Human and murine ApoE markedly alters A beta metabolism before and after plaque formation in a mouse model of Alzheimer"s disease. Neurobiol. Dis. 9(3):305-318. This paper discusses analyzing beta amyloid by ELISA. This paper has an interesting variation on the measurement of beta amyloid: they looked at soluble beta amyloid and also insoluble beta amyloid. Here are the key points of their protocol: For analysis of total beta amyloid levels, half of the hippocampus from each animal was Dounce homogenized in 5 M guandine (50 nM Tris-HCl, pH 8.0) and beta amyloid ELISA was performed as described previously (Johnson-Wood, et al. 1997). For analysis of soluble beta amyloid, the other half of the hippocampus was homogenized on ice in 400 microliters TBS (25 mM Tris-HCl, 150 mM NaCl, 3 mM EDTA, pH 7.4) containing protease inhibitors (20 micrograms/mL aprotinin, and 10 micrograms/mL leupeptin). Homogenates were spun at 125,000xg in polyallomer tubes in a Sorvall RP100 AT4-406 rotor for 1 hour at 4 degrees C and levels of beta amyloid total in the resultant supernatant (defines as soluble Abeta total) were obtained by beta amyloid ELISA. Percentage of soluble Abeta total was defined as the soluble (TBS-extractable) value divided by the total tissue (guanidine-extractable) value.
For Brain Tissue Homogenization, Prepare the Following Solutions: 5 M guanidine HCl 50 mM TrisHCl, pH 8.0. Reaction Buffer BSAT-DPBS (Dulbecco’s phosphate buffered saline with 5% BSA and 0.03% Tween-20, see formulation below) supplemented with 1x Protease Inhibitor Cocktail from Calbiochem (catalog code 539131; contains AEBSF, aprotinin, E64, EDTA, and leupeptin). BSAT-DPBS Formulation 0.2 g/L KCl 0.2 g/L KH2PO4 8.0 g/L NaCl 1.150 g/L Na2HPO4 5% BSA 0.03% Tween-20 to 1 L ultrapure water and adjust the pH to 7.4. Protocol: Determine the wet mass of the mouse hemibrain (100 mg) or a human brain sample in an Eppendorf tube (Fisher K749520-0000). Add 8 x mass of cold 5 M guanidineHCl / 50 mM Tris-HCl (Solution "A", above) to the tube by 50 - 100 µL aliquots and grind thoroughly with a hand-held motor (Fisher K749540-0000) after each addition. (Optional: transfer the homogenate from above to 1 mL Dounce homogenizer and homogenize thoroughly.) Mix the homogenate at room temperature for 3 - 4 hours. The sample is stable and can be freeze-thawed many times at this stage. Dilute the sample with cold Reaction Buffer (Solution "B"). Centrifuge (microfuge or Sorvall) at 16,000 x g for 20 minutes at 4°C. Save the supernatant for the assay.
