Preparation of Protein Lysates from Mouse Tissues: Difference between revisions
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added different RIPA volume for WAT |
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#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. | #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. | #Cool the centrifuge to 4C. | ||
#Add 20 uL/mg of RIPA (keep on ice) or other buffer to tissue (400-1000 uL) | #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). | #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. | #Remove stainless steel bead or transfer the homogenized tissue to a new tube. Keep tissue on ice. | ||