Set-Up: - Lysis Buffer (P Buffer)
- dounch homogenizers (tight)
- 1X PBS (cold)
- cell scrapers
- eppendorf tubes (1.5 ml)
- centrifuge tubes (1.5 mL, round top, special order)
***All steps and solutions should be kept on ice***
Protocol: - Aspirate media from cells
- Rinse 1X with PBS
- Add 500 µL of Lysis buffer to plate and scrape cells.
- Pipet plate content into dounce homogenizer and dounce 30X. (Strong popping sound equals good douncing)
- Pour solution into 1.5 mL tubes and centrifuge at 3000 rpm for 10 minutes at 4 degrees C.
- Transfer supernatant into centrifuge tubes and freeze until final steps.
Once all samples have been collected: - Ultracentrifuge supernatant at 30,000 rpm for 90 min at 4 degrees C
- Transfer supernatant to a new eppendorf tube and Resuspend pellet in 150 µL Lysis buffer with 0.1% SDS added. (Resuspend by sonicating for 10 s)
- Measure Protein Concentrations (BioRad Assay) for both supernatant and pellet
- Run samples on Western Gel and use mB-catenin Antibody from Transduction Laboratories (1:1000)
Previous Concentrations have been: 30 µg cytosolic protein (supernatant) 15 µg membrane protein (pellet)
Lysis Buffer (Modified from original): | | Stock | for 20 mL | | 10 mM Tris (pH 7.5) | 1 M | 200 µL | | 140 mM NaCl | 5 M | 560 µL | | 5 mM EDTA | 250 mM | 400 µL | | 2 mM DTT | 100 mM | 400 µL | | PIC1 | | 40 µL | | PIC2 | | 40 µL | | Na-orthovanadate | | 200 µL |
Fill to volume with ddH2O
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