Normally I wash/detect with one primary/secondary-HRP antibody pair, strip, then wash/detect with the other primary/secondary-HRP pair which works well. However, I recently started working with a HRP-conjugated primary antibody for my protein of interest and wondered if I might add this together with the secondary antibody for my loading control to save the stripping and second washing.
Are there any general guidelines for Western blotting with multiple antibodies at the same time, i.e. changing antibody dilution?
An obvious first step is to make sure the HRP-conjugated primary isn't from the animal recognized by the secondary. I anticipate blotting with mouse HRP-conjugated anti-GFP and a secondary rabbit anti-mouse would not be a very good decision.