Rabbit anti fbw7

For immunoblotting, cells were seeded in 24 or 96 well plates (Corning) then later harvested by trypsinisation, centrifuged, washed and lysed in SDS buffer (0.35 M Tris pH 6.8, 0.1 g/ml sodium dodecyl sulfate, 93 mg/ml dithiothreitol, 30% glycerol, 50 μg/ml bromophenol blue). Proteins were resolved by SDS-PAGE then electro-blotted onto Immobilion-P membranes (Millipore). Membranes were blocked in 5% dried milk in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween-20) then incubated overnight at 4°C with the following primary antibodies diluted in milk: rabbit anti-Mcl-1 (Santa-Cruz), sheep anti-Tao1 [60 (link)], mouse anti-Cyclin B1 (Millipore), rabbit anti-Bcl-xL (Cell Signalling), sheep anti-Bub3 (Holland and Taylor, unpublished), rabbit anti-FBW7 (Bethyl), mouse anti-Bak (Calbiochem), rabbit anti-Bax (Santa-Cruz), mouse anti-myc tag (4A6, Millipore), rabbit anti-GFP (Cell Signalling). Following TBST washes, blots were incubated with appropriate horseradish-peroxidase-conjugated secondary antibodies (Zymed). Bound secondaries were then detected by addition of EZ-ECL Chemiluminescence Reagent (Biological Industries) or Luminata™ Forte Western HRP Substrate (Millipore) and imaged using a Biospectrum 500 imaging system (UVP).