Difference between revisions of "Preparation of Protein Lysates from Mouse Tissues"

Revision as of 13:07, 8 July 2009

Materials

• RIPA Buffer (see RIPA)
• Mouse Tissues

Protocol

1. Weigh frozen tissue samples, only need 100-300 mg of tissue. If there is too much cut it off and return the extra tissue to the -80
2. For fibrous tissues chop into small pieces with scissors
3. Add 3 volumes of RIPA to tissue and homogenize 20x with 1 mL glass homogenizer (normally on SHC bench)
4. Transfer lysate to fresh tube and keep on ice
5. Clean homogenizer and lyse remaining tissues
6. Centrifuge at 14 000 RPM at 4C for 10 min
7. Remove supernatant to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify
8. Do a bradford assay using 1 uL of clarified lysate and label tube with correct concentration.
9. Make 100 uL of SDS sample using 2X loading buffer
10. Snap freeze remaining clarified lysate and store at -80