Difference between revisions of "Preparation of Protein Lysates from Mouse Tissues"
From Bridges Lab Protocols
Davebridges (Talk | contribs) m (changed category) |
Davebridges (Talk | contribs) (updated protocol with new weights) |
||
| Line 1: | Line 1: | ||
==Materials== | ==Materials== | ||
| − | *RIPA Buffer (see [[Buffer/RIPA|RIPA]]) | + | *RIPA Buffer (see [[Buffer/RIPA|RIPA]]) or other Lysis buffer. Add protease inhibitors. |
| − | *Mouse Tissues | + | *Mouse Tissues (Frozen) |
==Protocol== | ==Protocol== | ||
| − | #Weigh frozen tissue samples, only need | + | #Cut frozen tissue on a glass plate on dry ice. Place in a new round bottom eppendorf tube. |
| − | + | #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. | |
| − | #Add | + | #Add 20 uL/mg of RIPA or other buffer to tissue (400-1000 uL) |
| − | # | + | #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). |
| − | + | ||
#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 (see [[Bradford Assay]]) | #Do a bradford assay using 1 uL of clarified lysate and label tube with correct concentration (see [[Bradford Assay]]) | ||
| − | # | + | #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 |
| − | #Snap freeze remaining clarified lysate and store at -80 | + | #Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80 |
Revision as of 16:11, 15 July 2013
Materials
- RIPA Buffer (see RIPA) or other Lysis buffer. Add protease inhibitors.
- Mouse Tissues (Frozen)
Protocol
- Cut frozen tissue on a glass plate on dry ice. Place in a new round bottom eppendorf tube.
- 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.
- Add 20 uL/mg of RIPA or other buffer to tissue (400-1000 uL)
- 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).
- 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)
- 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
- Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80
