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Assay for Free Cytosolic b-catenin

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:
  1. Aspirate media from cells
  2. Rinse 1X with PBS
  3. Add 500 µL of Lysis buffer to plate and scrape cells.
  4. Pipet plate content into dounce homogenizer and dounce 30X. (Strong popping sound equals good douncing)
  5. Pour solution into 1.5 mL tubes and centrifuge at 3000 rpm for 10 minutes at 4 degrees C. 
  6. Transfer supernatant into centrifuge tubes and freeze until final steps.

    Once all samples have been collected:
  7. Ultracentrifuge supernatant at 30,000 rpm for 90 min at 4 degrees C
  8. 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) 
  9. Measure Protein Concentrations (BioRad Assay) for both supernatant and pellet
  10. 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 M200 µL 
140 mM NaCl 5 M560 µL 
5 mM EDTA250 mM400 µL 
2 mM DTT100 mM400 µL 
PIC1  40 µL 
PIC2 40 µL 
Na-orthovanadate 200 µL 
Fill to volume with ddH2O