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Characteristics
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Spleen tissue 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, 5µg/mL of leupeptin. Tissue and cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. 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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