Adipose tissue lysis for western analysis
HNTG Lysis Buffer
5.96 g HEPES (50 mM)
4.38 g NaCl (150 mM)
50 mL glycerol (10%)
5 mL Triton X-100 (1%)
Bring to a volume of 400 mL using Milli-Q water and pH to 7.5 using NaOH. Bring to a total volume of 500 mL. Filter and store at 4 C. Add appropriate protease/phosphatase inhibitors before use.
Mince tissue, and add enough lysis buffer to cover tissue and homogenize with a dounce homogenizer 20 times.
Transfer to a 2.0 mL mircocetrifuge tube and rotate at 4 C for 30 min.
Centrifuge lysate at 18,000 x g , at 4 C, for 20 min.
Remove fat cake from the top, transfer the supernatant to a clean tube, discard pellet of debris and unlysed cells.
Protein concentration and denaturation
Determine concentration using the Bradford assay (Biorad reagent). Add 20-60 µg of protein to appropriate amount of 4X SDS loading buffer and boil for 5 min.