Rabbit anti dnp antibody

Following heat treatment, HT22 cells were lysed as explained above, 20 μg of total proteins were reduced by Laemmli-buffer and denatured at 95 °C for 5 min and applied to SDS-PAGE of 10% (w/v) acrylamide and followed by electrophoresis. The proteins for blotting were transferred onto a nitrocellulose membrane by Turbo Trans-blot system (Bio-Rad Laboratories). Following the electroblotting step, the membranes were equilibrated in TBS (100 mM Tris, 150 mM NaCl, pH 7.5) containing 20% methanol for 5 min, washed in 2N HCl for 5 min, incubated with 10 mM DNPH solution for 5 min, washed 3×5 min in 2N HCl and washed 5×5 min in 50% methanol. DNPH treated membrane was blocked with 5% non-fat dry milk in TBST (TBS contains Tween 20) for 1 h at room temperature with constant agitation. Blocked membrane was washed 3×5 min with TBST and incubated with rabbit anti-DNP antibody (Sigma) freshly diluted 1:2500 in 5% non-fat dry milk/TBST for 1 h at room temperature with constant agitation. Blotted membrane was washed 3×5 min with TBST and incubated with secondary anti-rabbit antibody (Bio-Rad) freshly diluted 1:5000 in 5% non-fat dry milk/TBST for 1 h at room temperature with constant agitation. The blotted membrane was washed 5×5 min with TBST. Membrane was developed using Lumi-Light western blotting substrate.