Preparation of Protein Lysates from Mouse Tissues: Difference between revisions
| Line 6: | Line 6: | ||
#Cut frozen tissue on a glass plate on dry ice. Place in a new round bottom eppendorf tube. | #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. | #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 20 uL/mg of RIPA or other buffer to tissue (400-1000 uL) | #Cool the centrifuge to 4C. | ||
#Add 20 uL/mg of RIPA (keep on ice) or other buffer to tissue (400-1000 uL) | |||
#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. | |||
#Centrifuge at 14 000 RPM at 4C for 10 min | #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 to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify | ||