Multiplexing is becoming “all the rage” these days. And why not? It’s an ideal technique for simultaneously detecting multiple proteins or protein species.
- Want to have an in-lane normalization control? – Multiplex it
- Want to quantify phosphorylation of your protein? – Multiplex it
- Need to detect 2 targets with the same molecular weight? – Multiplex it
- Don’t want to run multiple blots? – Multiplex it
- Want to have the most accurate quantitative results? – you guessed it,
But like all techniques, it helps to hear some wisdom from people who know what they are doing before you try it on your own.
Here are our favorite tips for multiplexing:
Don’t be chicken to mix the species
Here’s your chance to expand your antibody repertoire! You don’t want the same secondary antibody detecting both of your targets, so make sure your primary antibodies are made in different species of animals (or cells). Along with the usual suspects (anti-mouse, anti-rat, anti-goat), you can also buy more exotic secondary antibodies such as anti-guinea pig and anti-chicken.
Watch out for the relatives
Us animals are all related to one another. Therefore, sometimes your anti-mouse antibody might also recognize rat primary antibodies. To make sure this doesn’t foul up your results, use secondary antibodies that are highly cross-adsorbed against other species.
Try a kit
Want to take the guess work out of multiplexing? Use a kit, such as Advansta’s WesternBright MCF and MCF-IR. These kits come with all you need to detect two proteins simultaneously (you supply the primary antibodies).
Make sure your colors don’t overlap
Avoid colors whose emission spectras bleed into one another, otherwise you might have trouble telling who is who.
Try them alone first before getting them together
It is tempting to just jump right in, mix up your new antibodies and incubate away. But you will save yourself some aggravation if you take the time to titrate each antibody individually before you use them together.
Assign your colors carefully
You can make your multiplex blot better by carefully assigning the correct color to each target. Red is brighter than blue or green, so use this fluorophore for your targets that are expressed more weakly. Use blue or green for higher expressing targets.
Go further than you can see
You don’t have to limit yourself to traditional blue, green and red channels. New fluorophores extend the spectral range out of the visible range to the near infrared.
Secondary antibodies conjugated to near IR fluorphores have a higher signal-to-noise ratio increasing your sensitivity. They also have narrow emission spectra limiting cross-over between the dyes.
Just make sure you have an imager available with the proper excitation and emission capabilities.
These are our favorite tips for multiplexing, we’d love to hear yours.
Photo courtesy of Jynto.