Difference between revisions of "Preparation of Protein Lysates from Mouse Tissues"
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#Centrifuge at 14 000 RPM at 4C for 10 min | #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 | #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 | + | #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 | #Make 100 uL of SDS sample using 2X loading buffer | ||
#Snap freeze remaining clarified lysate and store at -80 | #Snap freeze remaining clarified lysate and store at -80 | ||
Revision as of 13:08, 8 July 2009
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
