Hi all,
This may be a really dumb question but I just want to clarify one thing when you scan your membrane for western blotting.
I have a primary antibody raised in mouse, so I used Alexa Fluor 488 goat anti-rabbit secondary antibodies. When I tried to scan the membrane using Odessey CLx membrane scanner, there are only two channels, 700 and 800nm, so I chose both of them and got really fate bands.
Is this because I used Alexa 488 secondary antibodies? If I had used other Alexa fluor secondary antibodies such as 688 or 750, would I have got better results?
Yes, Alexa488 would not work very well with such long wavelength channels. You would want to have the excitation and emission spectra of the fluorophores matching that of the system.
One brief look at the CLx manual ( https://www.licor.com/documents/8prh6ps2abjbemx68412 ) reveals that the 700 nm (emission) channel uses a 685nm laser, and the 800nm (emission) channel uses a 785 nm laser. These would match very well with AlexaFluor 680 for 700 channel and AlexaFluor 790 for 800nm channel. Maybe there are other fluorophores that works best with this machine. You may want to ask others who have done experiment with CLx.
For a new imaging experiment, I always go to the Fluorescence SpectraViewer to look up what are the best hardware/fluorophore combinations:
https://www.thermofisher.com/nl/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html
Thanks Aeron for your useful info!
I will give it a go with AlexaFluor 800 for 800nm channel. Hope this one works better than 488.
Yinan,
Maybe it was just a typo but just to be sure: If your primary antibody has been raised in mouse, then you should use a secondary anti-mouse antibody (e.g. Alexa 6xx goat anti mouse).
Kaspar Gierke Thanks for pointing it out! Yes that was definitely a typo!
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