Following reports that experimental conditions may alter the expression of commonly used housekeeping proteins1 , leading journals now require researchers to use ‘total protein normalisation’ (TPN) methods when publishing data from quantitative Western blots. With TPN, the volume of the protein of interest is compared to the total amount of protein loaded in the lane. Instead of looking at the intensity of one protein, the total density for each lane is measured.
AzureRed is a novel fluorescent protein stain from Azure Biosystems optimised for TPN and Western blot quantification. Excited optimally with a 520nm (Cy3) laser light source or also UV light, AzureRed leaves NIR channels available for targets of interest. AzureRed can also be used in conjunction with chemiluminescence substrates.
AzureRed can be used on a range of imaging systems and is optimised for use with the Azure Q Module and analysed using AzureSpot software.
Benefits of using AzureRed
• Simple post transfer total protein normalisation stain
• Excited at 520 nm or UV leaving NIR channels free for target proteins
• Stain does not modify or alter proteins for true results
• Linear range over 3 orders of magnitude
• Methodology can detect <0.1 ug per lane
AzureRed enables total protein normalisation whilst leaving NIR channels available for targets of interest
A membrane was stained with AzureRed (gray) and probed for three different proteins using AzureSpectra secondary antibodies – AzureSpectra 490 – blue, AzureSpectra 700 – red, AzureSpectra 800 – green. The resulting fluorescent Western blot was scanned on a Sapphire Biomolecular Imager to detect all three proteins and the total protein stain simultaneously.
Material available for download
AzureRed fluorescent protein stain brochure
Application note: Accurate Western blot normalisation with AzureRed fluorescent protein stain
AzureRed total protein membrane staining protocol
1. R. E. Ferguson, H. P. Carroll, A. Harris, E. R. Maher, P. J. Selby, R. E. Banks, Proteomics 2005, 5, 566
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