Tissue Lysate Preparation

Lysate preparation:

1. Remove tissue/organ of interest from the animal and flash freeze in liquid nitrogen. Store the tissue/organ at -80°C until further use.
2. Suspend the frozen tissue in 5 volumes ice-cold lysis buffer [50 mM Tris pH 7.4, 5 mM EDTA pH 8, 1% Triton X-100 and protease inhibitor cocktail (Complete, EDTA-free, Roche)].
3. Homogenize the tissue with a polytron homogenizer. Centrifuge homogenates1 hr at 100,000 x g, 4°C.
4. Transfer the supernatant to a clean tube and measure protein concentration using the Bradford method. Adjust protein concentration to 3 mg/ml with lysis buffer. Store lysates at -80°C until further use.