Multiplex Western Blots: 6 Strategies that Work

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Are you having problems with your multiplex fluorescent Western blots? In this post, we’ll cover the exact protocols in more detail in some of our application notes that cover phosphorylation/total protein detection, and single step normalization.

Detecting one protein, two proteins, three proteins? How about FOUR proteins on the same Western Blot? Multiplexing your Western blots allows you to save money on each experiment.

Here at Azure Biosystems we’re great believers in the power of fluorescent multiplexing in Western blots, which has driven a lot of the development of our line of Azure imagers. But we recognize that taking the step from a standard HRP/chemiluminescent-based approach can be daunting. Keep reading for 6 tips to make the switch as easy as possible.

Azure fluorescence capabilities vs. competitor fluorescence, 3 proteins, plus total protein staining, and multiplex fluorescent Western blot

Browse our 6 multiplex Western blot expert tips

AZURE EXPERT TIP #1: Increase your concentrations

The concentration of both the primary and secondary antibody required may be increased compared to chemiluminescent Westerns depending which imager you’re using. In more modern or laser-based imagers, this effect may be less marked.

AZURE EXPERT TIP #2: Test individually first

Going hand in hand with our first tip, it always makes sense to test your antibodies individually first. That way you can determine optimal concentrations, factors contributing to background or lack of specificity in a simpler environment, rather than try to unpick 3 different primary and secondary antibodies at once.

AZURE EXPERT TIP #3: Use adsorbed secondary antibodies

Although it sounds simple, many people don’t consider that they are now adding multiple antibodies from multiple species onto a single blot. If you work with multiple fluorescence in other fields you’ll already be aware of the importance of cross-adsorbing secondary antibodies to reduce inter-species cross reactivity. But many people don’t consider this for Westerns, it’s worth checking out your antibodies to ensure they meet the grade.

Multiplex fluorescent Western blot
Multiplex fluorescent Western blot imaged with an Azure Imager using Cy3 and Cy5.

AZURE EXPERT TIP #4: Expand the spectrum with multiplexing

Three-color Western blots are exciting, but what about five colors? With near-infrared capability, the number of spectrally distinct peaks that can be isolated can be increased greatly. Obviously, this may require a bit of work up to get the antibodies optimized, but imagine the time and cost savings generated by performing one Western for five proteins.

The Sapphire Biomolecular Imager is an imager capable of detecting up to four proteins on the same Western blot, which allows you to save money on reagents each time you do an experiment. It uses 4 fluorescence channels to easily quantify overlapping bands. Read more on multiplex protein detection here

Since the release of this blog post, the Azure Sapphire has been succeeded by the new Azure Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins.

AZURE EXPERT TIP #5: Check your membrane

Some membranes will auto-fluoresce when exposed to UV light generating a high background signal, although more and more fluorescent safe membranes are being developed. We would also recommend switching to using PVDF membranes from nitrocellulose due to its increased sensitivity, as we discussed previously.

Package of pre-cut PVDF membranes for Western blotting applications
PVDF membranes reduce background noise for improved sensitivity.

AZURE EXPERT TIP #6: Choose the right channel for your protein

Unfortunately all detection channels were not created equally. For standard fluorescence use blue to detect your highest abundance protein, green the middle and red for your lowest abundance protein. If introducing NIR the excellent sensitivity and low background achieved with these fluorophores also makes them ideal for low abundance proteins.

Want to read more? Check out this paper from the National Institute of Health that utilizes multiplex Western blots using microchip electrophoresis.

Have more questions or want to learn more about multiplexing and how it can improve the way you research? Fill out the form on the left- we’ll be in touch.

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