Every datasheet recommends a starting dilution, but the dilution that works in our hands isn’t always the dilution that works in yours — sample preparation, blocking, and detection chemistry all change the optimal range. Here’s how we recommend titrating.
Western blot starting protocol
For a new rabbit polyclonal antibody on a new sample type, start with three dilutions in parallel on the same blot: 1:500, 1:1000, and 1:2000. Run identical sample lanes on each lane. Use the same secondary, same exposure, same everything — only the primary varies.
Score for two things: signal intensity at the expected molecular weight, and background. The best dilution is the one with the cleanest signal-to-noise ratio, not the one with the strongest absolute signal.
Common mistakes
Under-diluting usually causes non-specific bands and high background. If you’re seeing many bands at unexpected sizes, try a more dilute concentration before assuming the antibody is non-specific.
Over-diluting shows up as weak or absent signal at the expected band. If you don’t see any signal at 1:2000, drop back to 1:500 before assuming the target is absent.
For IHC and IF
IHC on FFPE tissue typically tolerates much more concentrated antibody than WB — start at 1:100 to 1:200 for IHC. IF on cultured cells sits in between, around 1:500.