Difference between revisions of "Preparation of Protein Lysates from Mouse Tissues"
From Bridges Lab Protocols
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#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. | #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. | ||
#Cool the centrifuge to 4C. | #Cool the centrifuge to 4C. | ||
| − | #Add 20 uL/mg of RIPA (keep on ice) or other buffer to tissue (400-1000 uL) | + | #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. |
#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). | #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). | ||
| − | #Remove stainless steel bead or transfer the homogenized tissue to a new tube. | + | #Remove stainless steel bead or transfer the homogenized tissue to a new tube. Keep tissue on ice. |
#Centrifuge at 14 000 RPM at 4C for 10 min | #Centrifuge at 14 000 RPM at 4C for 10 min | ||
#Remove supernatant and place into clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify | #Remove supernatant and place into clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify | ||
#Follow Protein Lysate instructions for Bradford Assay (see [[Bradford Assay]]) | #Follow Protein Lysate instructions for Bradford Assay (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 | #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 | ||
| − | #Heat samples with loading buffer at | + | #Heat samples with loading buffer at 85C for 2 mins |
#Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80 | #Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80 | ||
Latest revision as of 16:06, 25 April 2019
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.
- Cool the centrifuge to 4C.
- 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.
- 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).
- Remove stainless steel bead or transfer the homogenized tissue to a new tube. Keep tissue on ice.
- Centrifuge at 14 000 RPM at 4C for 10 min
- Remove supernatant and place into clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify
- Follow Protein Lysate instructions for Bradford Assay (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
- Heat samples with loading buffer at 85C for 2 mins
- Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80
