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: assets.thermof...
- 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: www.thermofish...
more on western blotting: bit.ly/westernb... ; KZitem: • Western blots: the wha... & more practical lab tips and tricks here: bit.ly/lab_tri...
blog form: bit.ly/western...
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbioc...
Негізгі бет Probing a western blot membrane for multiple things: multiplexing, stripping & re-probing, etc.
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