Description:
SW1353 lysate was prepared by homogenization in modified RIPA buffer (150 mM
sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylenediaminetetraacetic acid, 1 mM
phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1%
sodium dodecylsulfate, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin. Cell debris was
removed by centrifugation. Protein concentration was determined with Bio-Rad protein
assay. The SW1353 lysate was boiled for 5 min in 1 x SDS sample buffer (50 mM TrisHCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue)
containing 50 mM DTT.