Hey everyone, I’ve been working on a few experiments that require antibody labeling, and I’ve run into some confusion I was hoping to clarify here. Specifically, do we always need to use both primary and secondary antibodies in immunoassays like Western blot or immunofluorescence? Or is there any scenario where the primary alone is sufficient?

I understand the general workflow, but I’ve come across some resources that suggest alternatives. Still, I don’t want to cut corners if using both antibodies is critical for signal clarity or specificity. Can anyone share some insights on whether secondary antibodies are always necessary or if certain types of primary antibodies can eliminate that step? I’d appreciate hearing how others approach this in their protocols.