Sometimes black and white is fine. In fact, wouldn’t it be nice if all your results were black and white?
But sometimes, you need to go full color to get the data you need. Giving you the ability to multiplex and detect two or more proteins simultaneously (even overlapping ones!), fluorescent Westerns can be a powerful tool.
They are most useful for:
- Detection of 2 proteins simultaneously for in-lane normalization
- Easy visualization of loading controls
- Studying posttranslational modifications
- And fluorescent Western blotting is the best method for quantitative Westerns
Whether you are new to fluorescent Westerns or just need to optimize your experiment, we have some useful fluorescent Western tips to help you color your world
Use a membrane with low autofluorescence
Many materials can autofluoresce – nitrocellulose is notorious for this. To cut down on your background, use a low fluorescence PVDF membrane designed for fluorescent Westerns.
Avoid dyes and ink
Membranes are not the only things that can fluoresce. Pen ink and certain dyes, such as bromophenol blue, can fluoresce. To avoid random spots and everyone seeing your labeling system (Attempt #73 for publication quality blot), use pencil to label your blots and leave out fluorescent dyes from loading buffer – unless you can get away with running the dye off the gel and still see your protein.
Always use gloves
You don’t need to leave your mark in science by leaving fingerprints all over your blot. Make sure you handle the membrane with gloved-hands only.
Take some time to titrate your antibodies
More than likely you will need to use a little bit more antibody for a dazzling fluorescent Western. Estimates are that primary antibodies may need to be increased 2-5 fold. Make your life easier and just do the titration experiment first. This is the perfect time to try out the dot blotter!
If you are multiplexing, titrate each antibody individually to find the optimum concentration before using the two antibodies together.
Leave some space
If you are using fluorescently-labeled markers, you might want to skip a lane between the markers and your sample. This will keep the markers from overwhelming your samples.
Know when you need to keep things dark
You should store fluorescent antibodies in the dark. But you don’t need to use a darkroom when doing the actual experiment. Feel free to do your work out in the open on your lab bench. If archiving your blot after your experiment, keep it stored in the dark.
Choose your antibodies wisely
Avoid cross reactivity between your antibodies when multiplexing. Choose primary antibodies made in two different species of animals (the more distantly related, the better) and make sure your secondary antibodies are highly cross-adsorbed against other species.
Also make sure your fluorescent secondary antibodies have optically distinct spectra. You know, use red with green, or IR 700 with IR 800.
Blue signals tend to be weaker than green or red, so pair your highest expressing protein with an antibody in the blue channel. Likewise pair your low abundance protein with an antibody in the red channel.
Successful fluorescent Western blots are not magic; keep our tricks in mind for your own stunning results.
Photo courtesy of Dennis Skley
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