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Total Protein Normalization: Which Imager Should You Choose?

Total protein normalization (TPN) has become the preferred method for normalizing Western blot data. But to fully leverage its benefits, you need an imaging system that allows you to multiplex TPN with detection of your target proteins. Fortunately, we have just what you need! Whether you want to combine TPN with near infrared (NIR) fluorescent Western blotting, or with enhanced chemiluminescent (ECL) detection, read on to learn how our imaging systems enable you to do so with ease.

Why should you be using TPN?
A major advantage of TPN is that it delivers more accurate quantification of target analytes than the established practice of using individual housekeeping proteins. This is because TPN is less susceptible to change in response to experimental treatments, providing a more reliable baseline against which target protein expression can be compared. TPN also avoids the problem of over-saturation where low abundance analytes require high protein loads to reach the necessary sensitivity for detection since it has an incredibly broad dynamic range (1–50 μg of lysate).

How is TPN currently performed?
Current methods for TPN vary according to the chosen readout. Where TPN is combined with ECL detection, it is common practice to use specialized gels that chemically modify all of the proteins within each sample upon exposure of the gel to UV light, enabling their subsequent measurement. A drawback of this approach is that it has only a narrow range in which the protein load is linear.

In situations where TPN and NIR detection are paired, two distinct techniques are used. The first involves labeling the entire protein population of each sample with a fluorescent dye before loading the gel, a process that introduces an additional source of variability to the workflow. The second requires that the membrane be stained with a NIR reagent for TPN immediately after transfer; the membrane is then imaged and de-stained prior to NIR target detection. Because de-staining is never 100% complete, this latter method essentially restricts target detection to just one of the two available NIR channels.

How does our approach to TPN improve on existing methods?
Our range of imaging systems includes several models that allow target protein detection to be multiplexed with TPN – with no need for dedicated precast gels or laborious stripping and re-probing. Instead, you simply treat your blots with our total protein stain – TotalStain Q – between protein transfer and blocking, and process them as you would normally.

Which Azure imagers support TPN?
For TPN with NIR Western blot detection, the Azure 500Q and the Sapphire NIR-Q both provide detection of target analytes in the 700nm and 800nm channels, with a third (520nm) channel used to measure TotalStain Q. By reserving the NIR channels for your proteins of interest, sensitivity is uncompromised by integrating TPN into your Western blotting workflow.

Azure imaging systems enable target protein detection to be multiplexed with TPN. The Azure 300Q, Azure 500Q and the Sapphire NIR-Q all include the Q module (our optional green fluorescence channel) to quantify total protein staining.

Where TPN and ECL are performed in parallel, the Azure 300Q is a compact benchtop solution that can readily be upgraded to include visible and/or NIR fluorescent detection capabilities as your Western blotting requirements evolve. And if you already have an Azure 300 or Azure 500 in your lab, adding our optional green fluorescence channel – the Q module – to your system means you can easily begin multiplexing TPN without interruption to your Western blotting workflow.

If you’d like to learn more about how TPN can enhance your Western blotting data, take a look at our webinar.

Want to find out how our imaging systems enable you to multiplex total protein normalization with NIR fluorescent Western blot detection or ECL? Then check out our blog! We’re also giving away free samples of TotalStain Q – our newest reagent for total protein staining.