Western blot analysis
In order to detect casein in total cell extracts, cells (area 8 cm2) were washed in phosphate-buffered saline and lysed directly in 0.5 ml electrophoresis sample buffer (0.125 mol/l Tris HCI pH 6.8, 2% sodium dodecyl sulphate, 2% β-2-mercaptoethanol, 10% glycerol), shearing the DNA by repetitive pipetting, boiled for 10 min and stored at-20°C. The protein concentrations of the samples were determined using the BCA Protein Assay Reagent kit (Pierce and Warriner, Chester, UK).
One dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed as described by Laemmli [25] in 15% polyacrylamide gels with a 3% stacking gel. Proteins were transferred from gels to nitrocellulose filters (Schleicher and Schuell, London, UK) at a current of 0.8 mA/cm2 for 1 h using a semidry electroblotter [26]. After blocking nonspecific binding with 1% bovine serum albumin in phosphate-buffered saline/Tween (0.1%), the nitrocellulose was exposed to a polyclonal rabbit antimouse β-casein antibody diluted 1:10 000 in blocking solution. Primary antibodies were visualized by peroxidase-conjugated anti-lg antibodies and ECL detection reagents (Amersham).