Duodenum tumor tissue lysate was prepared by homogenization in lysis buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 microg/ml aprotinin, 5 microg/ml leupeptin, 1% Triton x-100, 1% DOC, 0.1% SDS). Tissue and cell debris was removed by centrifugation. The product was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% beta-mercaptoethanol.
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