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.

Tissue Lysis

• • 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.