Dear Nanthini,

what lysis buffer did you use? We prepared our brain samples according to:

"Human neocortex from aged controls, sporadic AD patients as well as different brain regions from APP-mutant patients were frozen in liquid nitrogen straight after sampling and stored at -80°C. Tissues were homogenized in ice-cold extraction buffer I containing 120 mM NaCl, 50 mM Tris-base (pH 8.0) supplemented with 1x complete protease inhibitor mixture (Roche Applied Science). After eight-low speed spins using dounce homogenizer, the homogenates were centrifuged 30 min at 17,000 x g at 4°C. The resulting pellets were re-homogenized in extraction buffer II containing 2% SDS (in aqua bidest.) supplemented with 1x complete protease inhibitor mixture using a Brandson Ultrasound Homogenizer. Homogenates were cleared by a second centrifugation at 17,000 x g for 30 min. The supernatants containing the insoluble fraction were aliquoted and stored at -20°C. "

All the best,

Vivek

The reaction between the antigen and primary antibody and primary with secondary antibody takes longer time to reach plateau sometimes. Thus sometimes incubating in both primary and secondary antibodies for a longer time than the usual 1h at room temperature or 24h at 4 degrees would yield better signal. See this article for more on this - http://www.sciencedirect.com/science/article/pii/S1056871911002528

But yeah there could be many things contributing to your no bands, thus looking up at the troubleshooting tips suggested by Henna is a good option too.