I have a simple western blot of CD31 in HUVEC lysate. With an anti-mouse-HRP secondary Ab I get a good signal/band. With fluorescent secondary antibody there is no signal. I have tried multiple secondary Abs and titrated their dilutions far down. HAs anyone had similar issues with fluorescent WB detection?
Our controls (grid or blotted secondsry Ab) work and I have also tried a cllaborator’s imager.
What protein concentration did you use? Chemiluminescence is much more sensitive and could explain why you’re able to detect CD31 but fluorescence is not working. Therefore, I’d recommend to increase the protein concentration for fluorescence detection. Good luck!
I agree with Dana, it's probably a matter of sensitivity. HRP produces signals from the substrate which are "collected" during the exposure time. Long exposure times needed might be an indicator for this.
If you really need fluorescent signals I have two ideas for you:
1. Use a fluorescent antibody against the species of your secondary antibody.
2. Use a biotinylated secondary antibody and fluorescent avidin in a 3-step-system.
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