How should I prepare my tissue extracts for IP?

While we do not have a general protocol for tissue-based lysate preparation, here are some helpful tips:
Freshly prepared lysates result in fewer nonspecific and degradation bands, and therefore yield a cleaner blot. In general, tissue extracts tend to contain more background bands and degradation products than cell line extracts. Using fresh, sonicated, and clarified tissue extracts may lessen background. Samples should always be lysed in appropriate buffers that include protease inhibitors and phosphatase inhibitors. Please see our recommended inhibitors in the protocol below. We recommend the use of Cell Lysis Buffer #9803 for best results by IP.
Below is our in-house protocol for preparing tissue extracts for IP:

1. Cut a small piece of tissue, approximately 3mm^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. Amounts can be adjusted based on how viscous the mixture is. We usually start with 500ul of Cell Lysis Buffer and add more if necessary.
4. Homogenize well (~20 strokes)
5. Sonicate at 50%, 3 times, for 10 seconds each or pass through a small gauge needle (21 gauge) multiple times if a sonicator is not available. Rest on ice in between sonicating for 10 seconds.
6. Microcentrifuge at 12,000 rpm for 15-20 minutes to remove insoluble debris.
7. Store supernatant at -80C before use.

Notes:
Keep unused tissue on dry ice.
Perform all steps on ice.
Tissue lysates have short shelf-lives and should be made fresh frequently.

Last updated: September 12, 2024