Ez ecl chemiluminescence reagent

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(link)], mouse anti-Cyclin B1 (Millipore), rabbit anti-Bcl-xL (Cell Signalling), sheep anti-Bub3 (Holland and Taylor, unpublished), rabbit anti-FBW7 (Bethyl), mouse anti-Bak (Calbiochem), rabbit anti-Bax (Santa-Cruz), mouse anti-myc tag (4A6, Millipore), rabbit anti-GFP (Cell Signalling).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).

Following SDS-PAGE, proteins were electroblotted onto Immobilon-P membranes. Following blocking in 5% dried skimmed milk (Marvel) dissolved in TBST (50 mM Tris pH 7.6, 150 mM NaCl, 0.1% Tween-20), membranes were incubated overnight at 4°C with the following primary antibodies diluted in TBST: 54H6 (Rabbit anti-Bcl-xL, 1 : 1000; Cell Signalling Technology), S-19 (Rabbit anti-Mcl-1, Santa Cruz Biotechnology), sheep anti-Tao1 (1 : 3000 [73 (link)]), rabbit anti-Bim (1 : 500; BD Biosciences), mouse anti-Bad (1 : 1000; Santa Cruz), rabbit anti-Bid (1 : 1000; Cell Signalling), mouse anti-Bax (1 : 1000; BD BioSciences), mouse anti-Bak (1 : 1000; Calbiochem), 4A6 (mouse anti-Myc tag; 1 : 1000; Millipore) and GFP (Rabbit anti-GFP; 1 : 1000; Cell Signalling). Membranes were then washed three times in TBST and incubated for at least 1 h with appropriate horseradish-peroxidase-conjugated secondary antibodies (Zymed). After washing in TBST, bound secondary antibodies were detected using either EZ-ECL Chemiluminescence Reagent (Biological Industries) or Luminata Forte Western HRP Substrate (Millipore) and a Biospectrum 500 imaging system (UVP).

Protein supernatants extracted from hypothalamus samples were run on 10% sodium dodecyl sulfate acrylamide gels and electro-blotted onto nitrocellulose membranes. The membranes were incubated with primary antibodies overnight at 4°C, followed by a second incubation with anti-rabbit or anti-mouse IgG. Primary antibodies specific for Sirt1, STAT3, phospho-STAT3, and total NFκB (Cell Signaling Technology, Beverly, MA, USA) were used. Finally, the EZ-ECL chemiluminescence reagent (Biological Industries, Cromwell, CT, USA) was added onto the membrane and bands were obtained.