Preparation of Protein Lysates from Mouse Tissues: Difference between revisions

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==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 100-300 mg of tissue.  If there is too much cut it off and return the extra tissue to the -80
#Cut frozen tissue on a glass plate on dry ice.  Place in a new round bottom eppendorf tube.
#For fibrous tissues chop into small pieces with scissors
#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 3 volumes of RIPA to tissue and homogenize 20x with 1 mL glass homogenizer (normally on SHC bench)
#Add 20 uL/mg of RIPA or other buffer to tissue (400-1000 uL)
#Transfer lysate to fresh tube and keep on ice
#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).
#Clean homogenizer and lyse remaining tissues
#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]])
#Make 100 uL of SDS sample using 2X 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
#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

  1. Cut frozen tissue on a glass plate on dry ice. Place in a new round bottom eppendorf tube.
  2. 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.
  3. Add 20 uL/mg of RIPA or other buffer to tissue (400-1000 uL)
  4. 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).
  5. Centrifuge at 14 000 RPM at 4C for 10 min
  6. Remove supernatant to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify
  7. Do a bradford assay using 1 uL of clarified lysate and label tube with correct concentration (see Bradford Assay)
  8. 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
  9. Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80
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