TITLE: Preparation of Brain Samples for LEGEND MAX™ Beta Amyloid ELISA
AUTHORS: Sam Li

[DESCRIPTION]
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[STEPS]
?. [Soluble and insoluble tissue fractionation - This protocol generates soluble and insoluble subcellular fractions for analysis of Aβ partitioning in tissues.]
Homogenize in TBS with protease inhibitors (Pierce sells excellent solid tablets for use with homogenization buffers) at 5mLs per 1g tissue. Teflon/glass homogenizer with 5-6 passes on ice. EDTA1 (2mM) can be a useful addition to homogenization buffers (see notes below).
?. [Soluble and insoluble tissue fractionation - This protocol generates soluble and insoluble subcellular fractions for analysis of Aβ partitioning in tissues.]
Spin for 20 minutes 350,000xg (or equivalent)
?. [Soluble and insoluble tissue fractionation - This protocol generates soluble and insoluble subcellular fractions for analysis of Aβ partitioning in tissues.]
Remove supernatant (S1 or soluble fraction) - usually contains
?. [Soluble and insoluble tissue fractionation - This protocol generates soluble and insoluble subcellular fractions for analysis of Aβ partitioning in tissues.]
Resuspend pellet (agitate to break up) with: (use only one method below)a. Detergent2 - 1% triton OR 0.6% SDS (see notes) in TBS/inhibitor buffer using same volume as homogenization step (15 minutes incubation).b. 70% Formic acid2, 40-50% of homogenization volume (incubate 30 minutes).
?. [Soluble and insoluble tissue fractionation - This protocol generates soluble and insoluble subcellular fractions for analysis of Aβ partitioning in tissues.]
Spin a second time (20 minutes x 350,000 g or equivalent) and remove supernatant - detergent or formic acid extract fraction (E1).
?. [Soluble and insoluble tissue fractionation - This protocol generates soluble and insoluble subcellular fractions for analysis of Aβ partitioning in tissues.]
Optional - if detergent extract was prepared above, then remaining pellet can be further extracted with formic acid - 70% formic acid, half homogenization volume, spin down 10 minutes in microfuge full speed (10 minutes x 14,000 g). Remove formic acid extract (E2).
?. [Soluble and insoluble tissue fractionation - This protocol generates soluble and insoluble subcellular fractions for analysis of Aβ partitioning in tissues.]
S1 and E1 fractions are best assayed immediately but can be stored at -20°C (including non-neutralized formic acid samples)
?. [Whole tissue extraction - This protocol generates a single fraction containing the total Aβ tissue load.]
Homogenize tissue as above in TBS with protease inhibitors and EDTA.
?. [Whole tissue extraction - This protocol generates a single fraction containing the total Aβ tissue load.]
To homogenate add concentrated Triton X-100, SDS (to final 1 or 0.6% respectively) or formic acid (to final 70%). Agitate by pipetting up and down to mix. Detergents and formic acid are added after tissue homogenization to reduce foaming and other potential problems.
?. [Whole tissue extraction - This protocol generates a single fraction containing the total Aβ tissue load.]
Spin for 20 minutes x 350,000 g (or equivalent).
?. [Whole tissue extraction - This protocol generates a single fraction containing the total Aβ tissue load.]
Remove supernatant for assay (whole brain extract).  NB High salt and biomolecule concentrations in formic acid whole tissue extracts can sometimes result in visible precipitants forming in later steps. These samples retain strong Aβ signal but we have not established to what degree signal attenuated under these conditions.