Preparation of Protein Lysates from Mouse Tissues: Difference between revisions

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 ==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==
-#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)
+#Cool the centrifuge to 4C.
-#Transfer lysate to fresh tube and keep on ice
+#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.
-#Clean homogenizer and lyse remaining tissues
+#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 to 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
-#Do a bradford assay using 1 uL of clarified lysate and label tube with correct concentration (see [[Bradford Assay]])
+#Follow Protein Lysate instructions for Bradford Assay (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
+#Heat samples with loading buffer at 85C for 2 mins
+#Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80