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Recombinant α- β- and γ-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays
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Chapters
- 00:00Title
- 00:33Preparation of Buffers and Reagents
- 00:38Preparing p-Nitrophenol Phosphate (pNPP) Buffer
- 01:21Preparing Malachite Green Solution
- 02:21Preparation of Activator
- 02:42Preparing Malachite Green + Tween 20 (MGT) Working Solution
- 02:58Preparing Threonine Phosphopeptide (pT)
- 03:31Synuclein Preparation
- 03:59Preparing Phosphate Standards
- 04:45Phosphate Standards Step 2
- 05:17PP2A Activity Assay
- 06:42Photometric Analysis
- 06:53Representative Results
- 09:01Conclusion
This publication presents a protocol showing how to measure the activity of recombinant protein phosphatase 2A catalytic subunit (PP2Ac) in response to recombinant α-synuclein, β-synuclein, or γ-synuclein proteins using a simple colorimetric assay. We show findings regarding α-synuclein specificity toward PP2Ac in mouse brain.
