Anti-MDA5 Antibody Works Great for Western Blot

To examine the expression levels of MDA5 in mouse brain and spleen, tissues were collected and lysed with the buffer [1% SDS, 10 mmol/L Tris-Cl (pH 7.6), 20 μg/μL aprotinin, 20 μg/μL leupeptin, and 1 mmol/L 4-(2-aminoethyl)benzenosulfonyl fluoride]. The protein concentrations were determined using the Bicinchoninic Acid Protein Assay kit (Pierce, Rockford, IL). Fiften micrograms of protein were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. After blocking, the membranes were incubated with the appropriately diluted primary antibody (1/1000) at 4°C overnight. After washing with TBST, the membranes were incubated with sheep anti-rabbit diluted HRP-conjugated secondary antibody (1/10000) at room temperature for 1 hour. Proteins were detected with the enhanced chemiluminescence kit (Amersham Pharmacia Biotechnology, Inc., Piscataway, NJ).