Optional Green Fluorescence Channel

Azure Biosystems Sapphire FL for total protein normalization

Add Total Protein Normalization to ECL or NIR Western Blots

Why Add a Fluorescent channel to Your Imager?

Issues you're facing

  • Publication guidelines are becoming increasingly strict: accurate quantitation of Western Blots requires normalization to an internal loading control.
  • Housekeeping proteins have poor linearity at high abundances.

The Best Solution: Enter the Q Module

Azure Biosystems provides a custom workflow for Total Protein Staining (TPS). The wider linearity and dynamic range of TPS is the new standard preferred by journals for accurate Western Blot quantification. We offer three imaging systems with optional fluorescent channels.

The Azure Q Module is an optional fluorescence channel intended for detecting TPS, such as TotalStain Q. With an extra channel, you are now able to detect up to two proteins of interest + total protein.

Imagers with fluorescent channels to detect total protein stains

Azure 300Q

Enjoy the sensitivity of chemiluminescence with the linearity and quantitative potential of fluorescent total protein stains, such as Azure TotalStain Q.

Normalize signal in your chemiluminescent western blots with the addition of a fluorescent channel. Detect more than just housekeeping proteins for quantitative western blots on top of your traditional ECL detection.

Azure 300Q imager with green channel for total protein normalization

Azure 500Q

Break through the preconceived limits of two fluorescent channels with the Azure Q module.

Near-infrared lasers provide the highest signal to noise ratio
with minimal crosstalk. The addition of a third fluorescent
channel to the camera-based imager allows the simultaneous
detection of housekeeping proteins or a total protein stain

Azure 500Q imager with green channel and 700nm and 800nm near-infrared lasers

New! Sapphire FL

Why limit your scanning capabilities to two channels?

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 Biosystems Sapphire FL for total protein normalization

TotalStain Q

Solves the issue of housekeeping proteins that lack linearity and normalization accuracy at high protein loads

TotalStain Q is a total protein staining reagent, compatible with chemiluminescent or near infrared fluorescent Western Blot detection. The linear range between 1–50 μg of lysate makes it ideal for normalization of low expressed proteins that require high loads of protein to reach the sensitivity for the detection of the target.

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

Significance of Western Blot Normalization​

Quantitative Western blots require the normalization of signal to an internal loading control. Guidelines from leading journals state that total protein normalization is preferred over normalization to housekeeping proteins.

Journal of Biological Chemistry
Transparency is the Key to Quality

“It is not uncommon to “correct” Western blot signals for protein loading by normalizing to [housekeeping proteins present in the same replicate], e.g.: β-actin, α-tubulin, transferrin, GAPDH, HPRT1. The problem with this approach is that a linear relationship between signal intensity and the mass or volume of sample loaded must be confirmed for every antigen… It is typically better to normalize Western blots using total protein loading as the denominator [given its wider linear range].”.

Fosang, Amanda J, and Roger J Colbran. “Transparency Is the Key to Quality.” The Journal of biological chemistry vol. 290,50 (2015): 29692-4. doi:10.1074/jbc.E115.000002

Journal of Proteomics
Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots

“Total protein staining (TPS) represents the actual loading amount more accurately than HKPs due to minor technical and biological variation. Further, the broad dynamic range of TPS solves the issue of HKPs that commonly fail to show loading differences above small loading amounts of 0.5-10 μg.”

Moritz CP. Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots. Proteomics. 2017 Oct;17(20). doi: 10.1002/pmic.201600189. PMID: 28941183

Documents Supporting Fluorescent Imaging

Document TypeDescription
Application NoteThree-Color Western Blots with AzureSpectra AntibodiesDOWNLOAD
Application NoteImaging Three-color Western Blots with the Azure c600DOWNLOAD
Application NoteAccurate Western Blot Normalization with AzureRed Fluorescent Protein StainDOWNLOAD
Application NotePhosphorylated Protein Detection is More Efficient by Fluorescent Western BlotDOWNLOAD
Application NoteIncreasing Assay Efficiency with Four Color DetectionDOWNLOAD
BrochureThe Azure Q ModuleDOWNLOAD
BrochureSapphire Applications OverviewDOWNLOAD
BrochureSapphire FL Biomolecular ImagerDOWNLOAD
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