903
edits
Changes
updated protocol with new weights
==Materials==
*RIPA Buffer (see [[Buffer/RIPA|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 10020-300 50 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. Record the weight of each tissue.#Add 3 volumes 20 uL/mg of RIPA or other buffer to tissue and homogenize 20x with 1 mL glass homogenizer (normally on SHC bench400-1000 uL)#Transfer lysate to fresh tube Add a stainless steel bead and keep tissues on ice#Clean homogenizer . Homogenize using Qiagen Tissue Lyser (3 min at 25Hz for WAT/Liver and lyse remaining tissues5 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]])
#Make 100 Prepare samples for gels by adding 800 ug protein to a final volume of 200 uL of lysis buffer. Add 200 uL of SDS sample using 2X loading bufferwith 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
