If you want to probe a western blot for multiple proteins (or modifications) you have a few options, depending on what you’re probing (how close together in size) and what you’re probing with (are your primary antibodies from the same or different species?)

• Multiplexing: if you have primary antibodies from different animals you can just use them both at the same time and prone with differently-labeled secondary antibodies 
• – when using fluorescent antibodies make sure they have non-overlapping wavelength absorption/emission
  • more here: https://assets.thermofisher.com/TFS-Assets/BID/Technical-Notes/fluorescent-western-blotting-guide-multiplexing-tech-note.pdf 
• If you have primary antibodies from the same organism, though, the secondary antibody would bind them both without distinguishing between them
  • if they’re far apart in size, you can cut the membrane between them (following prestained ladder bands) and probe them separately
    • but if they’re close in size that won’t work. This is a time that calls for stripping and reprobing. 
• For housekeeping proteins (loading controls) you can also buy conjugated primary antibodies and then probe with them separately 

here’s the recipe I used for stripping: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/stripping-reprobing-western-blots.html 

more on western blotting:  http://bit.ly/westernblotworkflow ; YouTube: https://youtu.be/Oun6_u6E090 & more practical lab tips and tricks here: https://bit.ly/lab_tricks_page     

more about all sorts of things:  #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0