TMB ELISA Peroxidase Substrate  

Rockland Immunochemicals produces a wide variety of buffers and substrates for use in ELISAs. Antigen was diluted in ELISA Microwell Coating Stabilizer (p/n MB-063-0100) added to the microwell plate and incubated overnight at 4°C. The plate was then blocked with ELISA Microwell Blocking Buffer with Stabilizer (p/n MB-064-1000) for 2 hours. The primary antibody was diluted in PBS Fish Gel Concentrate (1:10)(p/n MB-066-0100), added to the plate, and allowed to incubate 1 hour at room temperature. HRP conjugated secondary antibody was diluted in HRP Conjugate Stabilizer (p/n MB-060-0100), added to the plate, and allowed to incubate for 30 minutes at room temperature. TMB ELISA Peroxidase Substrate (p/n TMBE-1000) was added to the plate and allowed to incubate for 30 minutes at room temperature. The reaction was then stopped with 1M HCl and read at 450nm.