Changes

*RIPA Buffer (see [[Buffer/RIPA|RIPA]])or other Lysis buffer. Add protease inhibitors.*Mouse Tissues(Frozen)

#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. Record the weight of each tissue.#For fibrous tissues chop into small pieces with scissorsCool the centrifuge to 4C.#Add 3 volumes 20 uL/mg of RIPA (keep on ice) or other buffer to tissue and homogenize 20x with 1 mL glass homogenizer (normally on SHC bench400-1000 uL)for liver and muscle. Add 2-3uL/mg of RIPA for WAT.#Transfer lysate to fresh tube 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 tissuesRemove stainless steel bead or transfer the homogenized tissue to a new tube. Keep tissue on ice.

#Remove supernatant to 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 Follow Protein Lysate instructions for Bradford Assay (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#Heat samples with loading buffer at 85C for 2 mins#Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80