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).
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 an 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. Always make sure the total protein stain you use is compatible with the antibody binding and detection method. For the best TPN results, simply treat your blots with TotalStain Q, our total protein stain, 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.
HeLa lysate 10μg replicates imaged using TotalStain Q (Cy3 channel, green) and pSTAT3 on an Azure Sapphire Imager.
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.
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.
Azure imaging systems 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.
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 able to image both chemiluminescent and fluorescent signals and comes readily equipped with the 524nm laser to be able to detect TotalStain Q. The new Sapphire FL supports a broad range of excitation and emission wavelengths. We recommend using the standard 532 optical modules for detection of TotalStain Q
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.
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.
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