Transitioning from Chemiluminescent to Fluorescent Western Blotting
Do you regularly strip and re-probe your blots? Would you like a reliable way to quantify your bands? Consider adding a little color to your life with fluorescent Westerns. Fluorescent detection enables users to multiplex, which simplifies your workflow by eliminating stripping and re-probing. In addition, your data will be quantitative and the signal will be more stable over time. Here are some of our best tips to help you make the switch.
The Membrane Matters
Not all membranes are created equally! Choose a membrane with low autofluorescence so that your general background is low. This will help you achieve those great signal-to-background ratios that will give you the best limits of detection and quantification.
Filter your blocking and washing solutions to remove particulates. Fluorescent blots are more sensitive when it comes to particulates since they can stick to the membrane and cause speckled artifacts. These bright spots make accurate quantification difficult.
Handle with Care
Use powder-free gloves and only handle the membrane on its edges. Use blunt forceps that won’t scratch or damage the surface.
Dyes and ink are not your friends! Avoid using pens to mark the blot since the ink will likely fluoresce and cause background. Use a pencil if you absolutely need to annotate the blot. The bromophenol blue dye in your loading buffer also fluoresces so completely run the dye off the gel, otherwise cut it off before you transfer to the membrane.
Optimize Antibody and Sample Concentrations
It is not uncommon to require higher concentrations of sample and antibodies when fluorescent detection is used. When multiplexing, it is especially important to use pre-adsorbed antibodies to minimize undesirable cross-reactivity. If you plan on multiplexing, optimize the concentration of each sample, primary and secondary antibody combo independently. This is a great time to break out the dot blots to save time and money!
Choose the Best Antibody-Dye Conjugate for Your Protein
When multiplexing, it is important to use the longer wavelengths for the lowest abundance proteins in your sample. Short wavelengths often have elevated general background, so long exposures will make it difficult to detect your protein. If you decide that you need to use a NIR dye, increase the stringency of your wash buffer by adding SDS to the wash after the antibody-dye conjugate incubation step.
Spectradyes have the highest signal-to-background ratios after the blot has dried completely. Dip the blot in methanol for about 15 seconds then air dry before imaging. Then, place your blot on one of our Background Quenching Sheets to eliminate background fluorescence in the imaging environment caused by equipment contamination.
AdvanBlock-Fluor blocking solution:
SpectraDye Secondary Antibodies:
AdvanWash washing solution:
Background Quenching Sheets: