How should I prepare my tissue extracts for western blot?

Our tissue extracts are prepared as follows:

1. Cut a small piece of tissue (approximately 3 mm^3).
2. Place in a clean Dounce homogenizer.
3. Add 1mL cold 1X Cell Lysis Buffer #9803 + 1.0 mM PMSF #8553 + 1X Protease/Phosphatase Inhibitor Cocktail #5872.
4. Homogenize well (approximately 20 strokes).
5. Sonicate 3 times for 15 seconds each (cool on ice in between).
6. Microcentrifuge at 12,000 rpm for 15-20 minutes to remove insoluble debris.
7. 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.

Notes:

• Keep unused tissue on dry ice.
• Perform all steps on ice.
• 3X SDS Sample Buffer can be prepared using the Blue Loading Pack #7722 or Red Loading Pack #7723. To prepare fresh 3X Sample Buffer, add 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.
• Tissue lysates have short shelf-lives and should be made fresh frequently.

Last updated: February 26, 2024