Horseradish peroxidase hrp coupled secondary antibody

For the detection of p-AMPK, 15 μl of 1× PBS was added and animals were homogenized. Laemmli buffer (1×) was added, and the lysate was boiled at 99°C for 3 min to destroy all proteases. For the detection of Drp1-HA, 1× Laemmli buffer was added to the cytoplasmic and mitochondrial fraction, and the fractions were boiled at 99°C for 3 min. Electrophoresis was performed at 150 V in 1× SDS running buffer. Semidry blotting was performed to transfer the proteins onto a polyvinylidene difluoride membrane. Blotting was done at 100 V for 1.5 hours on a prior in methanol-activated membrane. The membrane was blocked in 5% milk in tris-buffered saline with Tween 20 (TBS-T) buffer (1 M) for 20 min followed by incubation with anti–p-AMPK (1:1000; Cell Signaling Technology), anti-HA (1:400; Roche), or anti-Creld (1:100; Davids Biotechnologie) antibody in 5% milk/TBS-T buffer at 4°C overnight. For immunodetection of the antibody, membrane was incubated with horseradish peroxidase (HRP)–coupled secondary antibody (1:7500; Santa Cruz Biotechnology) in 5% milk/TBS-T buffer in the dark at room temperature for 2 hours. Detection of antibodies was done with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) incubated for 1 min and exposed for 30 s. Signal acquisition was performed with Curix 60 (AGFA).