Preparation of Protein Lysates from Mouse Tissues

Materials

• RIPA Buffer (see RIPA) or other Lysis buffer. Add protease inhibitors.
• Mouse Tissues (Frozen)

Protocol

1. Cut frozen tissue on a glass plate on dry ice. Place in a new round bottom eppendorf tube.
2. Weigh frozen tissue samples, only need 20-50 mg of tissue. If there is too much cut it off and return the extra tissue to the -80. Record the weight of each tissue.
3. Cool the centrifuge to 4C.
4. Add 20 uL/mg of RIPA (keep on ice) or other buffer to tissue (400-1000 uL) for liver and muscle. Add 2-3uL/mg of RIPA for WAT.
5. Add a stainless steel bead and keep tissues on ice. Homogenize using Qiagen Tissue Lyser (3 min at 25Hz for WAT/Liver and 5 min at 50 Hz for Muscle).
6. Remove stainless steel bead or transfer the homogenized tissue to a new tube. Keep tissue on ice.
7. Centrifuge at 14 000 RPM at 4C for 10 min
8. Remove supernatant and place into clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify
9. Follow Protein Lysate instructions for Bradford Assay (see Bradford Assay)
10. Prepare samples for gels by adding 800 ug protein to a final volume of 200 uL of lysis buffer. Add 200 uL of 2X loading buffer with B-ME to each lyaste. This will generate a 2 mg/mL protein solution in SDS Loading Buffer
11. Heat samples with loading buffer at 85C for 2 mins
12. Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80