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What factors should I consider when performing fluorescent western blots?
Posted September 13, 2024

Answer

Fluorescent Western blots use secondary antibodies conjugated to different dyes with non-overlapping spectral emissions to simultaneously detect multiple proteins. These are the most important factors to consider when performing fluorescent Western blots: 

  • Optimize the detection of each target separately before performing simultaneous detection when multiplexing. 
  • Titrate primary and secondary antibodies to determine the best concentrations for each. Use a bot blot and checkerboard titration for this purpose.  
  • Adjust antibody concentrations if necessary. You may need to increase primary antibodies to about 2-5 times higher than the concentrations used in chemiluminescent Westerns, while secondary antibodies may need to be adjusted to start at 1:5000 dilution. 
  • Use low-autofluorescence membranes such as PVDF (Polyvinylidene Fluoride). Avoid using nitrocellulose membranes that can cause high background signals. 
  • Use a pencil to mark the dot instead of inks or dyes such as Coomassie and bromophenol blue, which can autofluoresce.
  • To prevent cross recognition in multiplexing, use primary antibodies from different species and cross-adsorbed secondary antibodies  
  • Select fluorophores with distinct spectra to prevent overlap in multiplexing. 
  • Skip a lane between fluorescent molecular weight markers and samples when loading, to prevent signal bleed from the molecular weight markers into the sample lanes. 
  • Protect fluorescent antibodies by working with them on the bench but storing them in the dark. 
  • For better signal clarity, detect the strongest target in the blue channel, weakest in the red channel, and medium in the green channel. 
  • Store blots in the dark when archiving. 
  • Cleanliness is vital to preventing background noise. Clean all trays and equipment thoroughly before use. Use powder-free gloves to handle the gel and membrane and keep trays covered during incubation.