How should I prepare my tissue extracts for western blot?

General Considerations

When preparing tissue extracts for western blot analysis, it is important to handle samples carefully to prevent degradation.

• Temperature Control: Perform all steps on ice and keep any unused tissue on dry ice.
• Shelf-Life: Tissue lysates have short shelf-lives and should be made fresh frequently.

Tissue Extraction Protocol

Follow these standard steps to prepare your tissue extracts for downstream analysis.

1. 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.

Buffer Preparation Details

The loading buffer requires specific preparation steps before combining it with your prepared lysates.

• 3X SDS Sample Buffer: This 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 dithiothreitol (DTT) to 1 volume of 3X SDS loading buffer.

Last updated: June 29, 2026