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Preparation of Protein Lysates from Cells

161 bytes removed, 18:33, 15 December 2017
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#Incubate on ice for 15 minutes
#Centrifuge at 14 000 RPM at 4C for 10 min
#Remove supernatant to clean tube. If lysing fatcells, 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 160ul lysate, 40ul of 200 uL 10x reducing agent and 200ul of lysis 2x SDS sample bufferfor 400ul total volume. 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
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