164
edits
Changes
added different RIPA volume for WAT
#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 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]])
#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