Detailed protocol for mouse adipose tissue protein extraction

The Scherer lab, modified on 09/30/2019

Reagents:

1. RIPA buffer (50 mmol/L Tris-HCl (pH 8.0), 0.25 mol/L NaCl, 5 mmol/L EDTA)

2. Triton X-100

2. Protease inhibitor cocktail from Roche, #11697498001

3. Optional: phosphatase inhibitors if phosphorylated proteins will be measured

Equipment:

1. Qiagen TissueLyser II homogenizer

2. ThermoScientific microcentrifuge (temperature controlled)

Protocol:

1. Harvest adipose tissues and snap freeze in liquid nitrogen.

2. Add 1 mL RIPA buffer (~100mg adipose tissue) with protease inhibitor and homogenize using the TissueLyser II (Qiagen) at the highest frequency for 3-5 minutes until clear. Keep on ice.

3. Spin down at 6,000 RPM for 15 min at 4°C.

4. Carefully remove the fat cake and resuspend the loose pellet. (Alternatively, use

the pipette tip to penetrate the fat cake and transfer the solution and the pellet to

a new tube).

5. Add Triton X-100 to a final concentration of 1%.

6. Incubate at 4°C for 30-60 minutes.

7. Centrifuge at 12,000 RPM for 15 min at 4°C.

8. Transfer the supernatant to a new tube.

9. Store the extracted samples at -80°C until further BCA concentration measurement and Western Blotting.

Tips:

1. The initial volume of RIPA buffer can be added to 1.2 mL/100 mg tissue. Higher efficiency of depleting lipid will be achieved with larger volume of lysis buffer.

2. Step 8 and 9 can be repeated for twice to get rid of maximum amount of lipid.