The purpose of this experiment is not to quantitate colocalization, but rather to evaluate the presence and (if present) localization of a protein target. We have a really nice positive control antibody to prove that we are looking at what we claim we are looking at (infectious stages of the parasite C. parvum), but it's so bright (even at incredibly low dilutions) that it's nearly impossible to use alongside another antibody using the same exposure. The other antibody is clearly detecting infectious stages, but it requires higher exposure times to see it well. Thanks for the help!
I don't think there is a problem with this, as long as it just in order to see location.
Seems more than one possibility here including: 1) further dilution of your bright antibody; 2) different exposure times; however, you don't indicate whether the glow of the bright antibody might obscure your preferred target with longer exposure; and 3) use fluorescein for the bright antibody and photobleach it, while using a less bleachable fluorochrome for your target protein.
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