Why Are Differentially Expressed Proteins Identified by Proteomics Undetectable by Western Blot
Proteomics, particularly when employing techniques such as mass spectrometry, can identify differentially expressed proteins that are sometimes undetectable by Western blot. This discrepancy may arise due to several factors:
1. Differences in Detection Sensitivity
Proteomic techniques, such as mass spectrometry, typically offer higher sensitivity compared to Western blot, enabling the detection of low-abundance proteins that may fall below the detection threshold of Western blot.
2. Target Protein Specificity
Western blot relies on the binding specificity of antibodies to detect target proteins. If the antibody exhibits low specificity or poor affinity for the protein, or if there are quality issues with the antibody itself, detection failure may occur.
3. Post-Translational Modifications and Protein Isoforms
Proteins may undergo post-translational modifications or exist in alternative splicing isoforms, potentially altering their epitopes and impairing antibody recognition during Western blot analysis.
4. Resolution Differences Between Mass Spectrometry and Western Blot
Mass spectrometry provides higher resolution and can distinguish between closely related protein species, whereas Western blot lacks this level of discriminatory power.
Therefore, when differentially expressed proteins identified by proteomics cannot be detected by Western blot, it is essential to consider these contributing factors. Optimization of experimental conditions or the use of alternative validation methods may be necessary to resolve such inconsistencies.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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