Preparation of Protein Lysates from Mouse Tissues: Difference between revisions
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#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) | ||
#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. | #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 | #Centrifuge at 14 000 RPM at 4C for 10 min | ||
#Remove supernatant and place into clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify | #Remove supernatant and place into clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify | ||
#Follow Protein Lysate instructions for Bradford Assay (see [[Bradford Assay]]) | #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 | #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 | #Heat samples with loading buffer at 85C for 2 mins | ||
#Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80 | #Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80 | ||