Robust Quantitation for Western Blots Using Total Protein Normalization (TPN)
Western blotting is a staple of molecular biology, enabling researchers to identify protein biomarkers and signaling changes that drive various diseases. However, meeting the increasingly stringent quantitative and reproducibility standards demanded by high-impact journals has become a challenge.
In this Teach Me in 10 episode, Phil Beckett, discovery imaging field application specialist at Cytiva, explains how to optimize Western blot workflows using total protein normalization (TPN), imaging, and detection strategies.
Watch this episode to explore how to:
- Improve accuracy and reproducibility with in‑lane and total protein normalization
- Enable wider dynamic range, stable signals, and quantitative confidence
- Generate robust, publishable Western blot data for high-impact journals
Further Resources:
- Amersham™ ImageQuant™ 800 Western Blot Imaging Systems: Flexible gel and blot imaging platform that allows you to easily image low and high abundance proteins automatically.
- ImageQuant™ TL Analysis Software: Analysis software for gel and blot images, multiwell plates, and colony counting applications.
- Cy5 Total Protein Normalization in Western Blot Analysis: The publication shows that using Cy5‑prelabeled total protein as a normalization control in Western blotting is more accurate and reproducible than traditional housekeeping proteins, which can vary in expression and introduce quantification errors.
- Amersham™ QuickStain Protein Labeling Kit: Amersham QuickStain enables protein detection directly after SDS-PAGE or transfer. Supports Western blot normalization for quantitative Western blotting.
- Amersham™ CyDye DIGE Fluors: CyDye DIGE Fluor minimal dyes and saturation dyes are size- and charge-matched fluorescent dyes specifically designed for detecting protein abundance differences in 2-D Fluorescence Difference Gel Electrophoresis using Ettan DIGE system.
- Request a demo: Try the Amersham™ ImageQuant™ 800 Western blot imaging systems in your own lab with your own samples.
Meet the Author
Assistant Science Writer
Interviewing
Discovery Imaging Field Application Specialist
