Spleen tissue lysate (7 Days Old) was prepared by homogenisation 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.
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% β-mercaptoethanol.