May 10, 2024
Protein Extraction for Amyloid Beta Fractionation
- Lisa Blackmer-Raynolds1,
- Tim Sampson1
- 1Emory University
Protocol Citation: Lisa Blackmer-Raynolds, Tim Sampson 2024. Protein Extraction for Amyloid Beta Fractionation. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8ky1l5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 08, 2024
Last Modified: May 10, 2024
Protocol Integer ID: 99473
Keywords: amyloid beta, fractionation, solubility, protein
Funders Acknowledgement:
NIH
Grant ID: F31AG076332-02
ASAP
Grant ID: ASAP-020527
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.
Preparation
Preparation
5x Homogenization Buffer (store 4oC):
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) **Caution Corrosive**
Dilute 5x Homogenization buffer to 1.5x in MiliQ Water and pH to 14
Tissue Processing
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