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V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting
Published on: December 30, 2013
Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting
Samantha L Eaton1, Sarah L Roche, Maica Llavero Hurtado
1Division of Neurobiology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom.
Plos One
|September 12, 2013
Summary
Common protein loading controls like actin are often differentially expressed in disease models, skewing quantitative Western blot results. Total protein analysis offers a more reliable normalization method for accurate protein expression studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Western blotting is a crucial technique for protein expression analysis.
- Quantitative Western blotting (QWB) advancements enable high-resolution pathway analysis.
- Differential expression of reference proteins poses a challenge for accurate QWB.
Purpose of the Study:
- To investigate the differential expression of common loading controls in various tissues and disease models.
- To highlight the impact of using unreliable loading controls on QWB data.
- To propose a more robust normalization method for QWB.
Main Methods:
- Quantitative Western blotting (QWB) was performed on tissues from animal models of neurodegeneration and other conditions.
- Analysis of published "-omics" data to assess loading control expression.
- Total protein analysis using stains like Coomassie blue was employed as an alternative normalization strategy.
Main Results:
- Common loading controls (actin, tubulin) were found to be differentially expressed in animal models of neurodegeneration.
- Differential expression was observed across multiple tissues, including nervous system, bone, fat, and internal organs.
- Normalizing to beta-actin in a Spinal Muscular Atrophy model skewed data by approximately 20%.
Conclusions:
- Commonly used single protein loading controls are often unreliable for quantitative Western blotting.
- Differential expression of loading controls can occur across various tissues and disease states.
- Total protein normalization provides a more robust and accurate approach for quantitative Western blotting.
