Difference between revisions of "Preparation of Protein Lysates from Mouse Tissues"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (added categories) |
Davebridges (Talk | contribs) m (changed category) |
||
| Line 18: | Line 18: | ||
[[Category:Protein]] | [[Category:Protein]] | ||
[[Category:SDS-PAGE]] | [[Category:SDS-PAGE]] | ||
| − | [[Category:Mouse]] | + | [[Category:Mouse Work]] |
[[Category:Tissues]] | [[Category:Tissues]] | ||
Revision as of 14:42, 28 May 2012
Materials
- RIPA Buffer (see RIPA)
- Mouse Tissues
Protocol
- 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
- For fibrous tissues chop into small pieces with scissors
- Add 3 volumes of RIPA to tissue and homogenize 20x with 1 mL glass homogenizer (normally on SHC bench)
- Transfer lysate to fresh tube and keep on ice
- Clean homogenizer and lyse remaining tissues
- Centrifuge at 14 000 RPM at 4C for 10 min
- Remove supernatant to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify
- Do a bradford assay using 1 uL of clarified lysate and label tube with correct concentration (see Bradford Assay)
- Make 100 uL of SDS sample using 2X loading buffer
- Snap freeze remaining clarified lysate and store at -80
