We want to extract Amyloid Beta from cultured astrocytes.

We came across a protocol by Kienlen Campard et al from 2002 used for Amyloid Beta quantification in ELISA:

1. Neurons (approximately 10^7) were scraped in ice cold PBS.

2. Cell pellets were solubilized in 300 microliters of 70% formic acid. 3. 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.

4. 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. 5. Protein concentration was determined by the BCA assay. 6. 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).

7. Beta amyloid 1-40 and Beta amyloid 1-42 concentrations were determined by ELISA using 100 microliters of neutralized extract.

My specific questions are:

1. What is the alkaline carbonate buffer used for? does it pull down any additional unwanted molecules in the last centrifugation?

2. Why a neutralization step done to the supernatant before the ELISA? is it again to remove any unwanted molecules?

Am I right to assume this is the case due to Amyloid Beta's "stickiness"?

Thank you for any help.