Add Total Protein Normalization to ECL or NIR Western Blots
Publication guidelines are becoming increasingly strict: accurate quantitation of Western Blots requires normalization to an internal loading control. One issue is that housekeeping proteins have poor linearity at high abundances. OUR SOLUTION? A custom workflow for Total Protein Staining (TPS).
The Azure Q Module is an optional fluorescence channel intended for detecting TPS such as TotalStain Q. With an extra channel, you can still detect up to two proteins of interest + total protein.
The wider linearity and dynamic range of TPS is the new standard preferred by journals for accurate Western Blot quantification.
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.
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
With the Sapphire NIR-Q scanner, target two proteins of
interest in the 700 and 800nm channels and add a third target
protein OR total protein stain using the Q module (520nm).
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.
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
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