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MAFA is a phosphoprotein. The multiple phosphorylation sites on this protein cause a mobility shift, resulting in the protein migrating slower by gel electrophoresis. This has been confirmed by treatment with CIAP, which has been shown to result in a shift in MAFA’s mobility from approximately 48 kDa to a more rapidly migrating form of approximately 40 kDa (see PMID: 17682063 Figure 1A; https://pubmed.ncbi.nlm.nih.gov/17682063/).
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Our tissue extracts are prepared as follows:
- Cut a small piece of tissue (approximately 3 mm^3).
- Place in a clean Dounce homogenizer.
- Add 1mL cold 1X Cell Lysis Buffer #9803 + 1.0 mM PMSF #8553 + 1X Protease/Phosphatase Inhibitor Cocktail #5872.
- Homogenize well (approximately 20 strokes).
- Sonicate 3 times for 15 seconds each (cool on ice in between).
- Microcentrifuge at 12,000 rpm for 15-20 minutes to remove insoluble debris.
- Add 333uL of 3X SDS Sampler Buffer (adjust as needed depending on how much 1X Cell Lysis Buffer you added) or store at -80C in 1X Cell Lysis Buffer.
- Keep unused tissue on dry ice. …