Most secondary antibodies are used in low concentrations typically between 1 and 10 µg/mL. Within that range, a good starting concentration for a typical secondary antibody would be a dilution of 1:1,000. This low concentration offers optimal performance with minimal background interference. Higher concentrations result in high background noise from too much non-specific binding.

If your staining is extremely bright or the reading shows too much background at a 1:1,000 dilution, consider using a higher dilution from 1:2,000 to 1:10,000.