Protein Extraction for Amyloid Beta Fractionation
Tim Sampson, Lisa Blackmer-Raynolds
Abstract
This protocol can be used to isolate protein from murine brain tissue (volumes listed are for hippocampal samples) by solubility. It creates three protein fractions: tris soluble, triton soluble, and formic acid soluble. This protocol can be helpful when evaluating amyloid beta pathology as different amyloid beta structures can be found within each fraction. For example, insoluble amyloid beta associated with amyloid plaques will be found within the formic acid soluble fraction, whereas more soluble non-plaque associated amyloid beta will be found within the other two fractions.
Steps
Preparation
5x Homogenization Buffer (store 4oC): oC):
125 ml of 1.0 M Tris
30 ml of 0.5 M MgCl2
25 ml of 0.1 M EDTA (pH to 7.2 and store at 4°C for up to 6 months)
20ml of MiliQ Water
1x Homogenization Buffer (dilute day of):
Dilute 5x Homogenization buffer to 1x in MiliQ Water and add 1 tablet protease inhibitor (cat#11697498001 Roche) per 10 mL
1x Homogenization Buffer with 1% Triton X (dilute day of):
Add 1% Triton X-100 to 1x Homogenization buffer made above
70% Formic Acid (store RT) Caution Acidic
7mL formic acid
3mL water
1.5x Homogenization Buffer pH 14 (store 4oC) oC) Caution Corrosive
Dilute 5x Homogenization buffer to 1.5x in MiliQ Water and pH to 14
Tissue Processing
Weigh frozen tissue sample and record for later normalization
Homogenize the tissue on ice using quick bursts of a sonicator in 400µl 1x Homogenization buffer
Centrifuge maximum speed at 4°C for 1hr
Collect supernatant as tris soluble fraction and store -80°C for later use
Homogenize the pellet on ice using quick bursts of a sonicator in 200µl 1x Homogenization Buffer with 1% Triton X-100
Centrifuge maximum speed at 4°C for 1hr
Collect supernatant as triton soluble fraction and store -80°C for later use
Homogenize the pellet on ice using quick bursts of a sonicator in 50µl 70% formic acid Be careful with acid
Centrifuge maximum speed at 4°C for 1hr
Collect supernatant as formic acid soluble fraction and neutralize with 1.5x Homogenization buffer pH14 on ice Be careful with acid and base*. Store -80°C for later use
