I have a matched antibody pair set for capturing my target, and both the capture and detection antibodies have been raised in rabbit. When I used an Anti-rabbit IgG as my fluorescent secondary antibody, I got a high fluorescent signal even when my target was not present (negative control) I think this is happening because both the capture and detection antibodies were raised in the same species.
I biotinylated my capture antibody in order to immobilize it to a Streptavidin-coated surface and the kit had the detection antibody already biotinylated. When I tried to use Streptavidin-PE as a fluorescent detection probe, I ran into the same problem where my negative controls had a really high fluorescent signal. I think the SA-PE is binding to my capture antibodies as well as the detection antibody and I am not able to distinguish a signal between my experimental conditions and my negative controls.
What could I do to troubleshoot this issue?
Use the unbiotinylated rabbit capture antibody to capture the antigen on a regular plate (not streptavidin-coated). Biotinylate the detection rabbit antibody. Use streptavidin-PE as the detection probe.
Or obtain a detection antibody that was raised in something other than a rabbit.
Adam is correct. You can easily playe bind the capture antibody to polystyrene plates if you use bicarbonate buffer over night. Typically 2 ug/ ml for capture works well
Adam and Lora are right, you should anti-Rabbit Antibody for detection. Like; Goat Anti-rabbit antibody, this should be labelled with either HRP, or any fluorophore and perform the detection accordingly.
Good Luck
- Badges
- Science topic
- Similar topics
- Cognitive Science
- Thinking