I use HRP-conjugated secondary Abs to detect my proteins. Sometimes I get no signal at all when using the imager. Strangely enough I see brown/yellow stains on my membrane where the protein should be detected. This occurs when i put more ore more sensitive Chemiluminescent Substrate on my Membrane. Doesn't this mean that the secondary Ab binds right an that the HRP "is working"? Why can't I detect the signal with the imager?
I sometimes also use a HRP-conjugated primary Ab for another protein and the detection works better.
Generally, if you can see the brown/yellow signal on you blot the signal will be very bright and everything is working. This also means that you likely don't need a long exposure time, but double check to ensure it is long enough to detect your signal.
What imager are you using? If it is a chemi-doc or something along those lines, make sure that the appropriate filter is being used to detect the emission of your HRP product.
Double check your dilutions are accurate. And use fresh blocking buffer.
Sometimes we see a brown signal on the membrane when the sample loading control is too high and antibody dilution is low. Try high dilution for primary antibody and less incubation time.
Most of the time, if you can see the brown/yellow signal on you blot the signal will be very bright and should be detected by the film/chemi doc.
There is something wrong with your detection technique. If you are using a film, make sure it is not exposed to light and if you are using the chemi-doc, then make sure your settings are correct (the selected filter can detect the emission of your HRP product).
What is wrong with my Western Blot detection?. Available from: https://www.researchgate.net/post/What_is_wrong_with_my_Western_Blot_detection [accessed Jun 13, 2017].
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