Preparation of Protein Lysates from Mouse Tissues: Difference between revisions
updated protocol with new weights |
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#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 | ||
# | #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 95C for 5 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 | ||