Which Imager is Best for Total Protein Normalization?

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Total protein normalization (TPN) is used to quantify the abundance of the protein of interest, without having to rely on housekeeping genes. It is usually done by incubating the membrane with a total protein stain. 
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 these Imaging Systems from Azure enable you to do so with ease.

Why you should use Total Protein Normalization (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).

HeLa-lysate-5μg-replicates-imaged-using-TotalStain-Q
HeLa lysate 10μg replicates imaged using TotalStain Q (Cy3 channel, green) and pSTAT3 on an Azure Sapphire Imager.

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 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 Azure's approach to TPN improve on existing methods?

Azure’s range of imaging systems includes several models that allow target protein detection to be multiplexed with TPN– without the need for dedicated precast gels, laborious stripping efforts, or re-probing to be done.

TotalStain Q PVDF for Western blotting from Azure Biosystems
TotalStain Q is an accurate and quantitative fluorescent total protein stain to perform normalization of Western blots imaged on both camera-based and laser-based instruments with green channel or Safe-Dyes channel.

For the best TPN results, treat your blots with a total protein stain like TotalStain Q between protein transfer and blocking, and process them as you would normally. TotalStain Q has a broad linear range, which makes it ideal for total protein normalization of low expressed proteins that require up to 50μg lysate/lane. Always make sure the total protein stain you use is compatible with the antibody binding and detection method.

Which imagers support TPN?

By reserving the NIR channels for your proteins of interest, sensitivity is uncompromised by integrating TPN into your Western blotting workflow. For TPN with NIR Western blot detection, using imaging systems such as the Azure 500Q, Azure 600, and the new Sapphire FL will provide detection of target analytes in the 700nm and 800nm channels. They also include a third channel used to measure TotalStain Q.

Azure Biosystems Sapphire FL for total protein normalization
The new Sapphire FL supports a broad range of excitation and emission wavelengths. We recommend the standard 532 optical module for detection of TotalStain Q.

The new Sapphire FL supports a broad range of excitation and emission wavelengths. We recommend using the standard 532 optical module for detection of TotalStain Q. Select Azure imaging systems also enable target protein detection to be multiplexed with TPN. The Azure 300Q and Azure 500Q both include the Q module (our optional green fluorescence channel) to quantify total protein staining. 

Azure 500Q imager with green channel and 700nm and 800nm near-infrared lasers
Shown: Azure 500Q imager with green channel and 700nm and 800nm near-infrared lasers. You are able to add a Q Module for Total Protein Normalization of NIR Western blots using TotalStain Q. The upgrade Azure 500Q will also detect one more fluorescent target.

In addition, the Azure 600 imager is also able to image both chemiluminescent and fluorescent signals. It comes readily equipped the 524nm laser to be able to detect TotalStain Q as well.

The Azure 600 Imager is a fully equipped laser scanning system capable of fluorescent, NIR, chemiluminescent, blue light, and UV imaging.

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.

Azure 300Q imager with green channel for total protein normalization
Add visible and/or NIR fluorescent detection capabilities as your needs evolve with the Q Module. The Azure 300Q imager is equipped with a green channel for total protein normalization.

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. It’s easy to do so, just contact us to upgrade your imager.

Want to find out how you can add multiplex total protein normalization with NIR fluorescent Western blot detection or ECL to your research? Send us a message using the form on this page. 

BONUS: We’re giving away free samples of TotalStain Q – our newest reagent for total protein staining! Grab a sample!

If you’d like to learn more about how TPN can enhance your Western blotting data, check out this webinar:

Let us show you just how easy getting good data can be. Fill out this form to be contacted by a life science expert today!

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