What Explains the Inconsistent Western Blot Detection of Recombinant Versus Tissue Proteins Using In-House Antibodies?
When self-produced antibodies yield a target band with recombinant proteins but fail to detect the target protein in tissue lysates via Western blotting, several factors may contribute to this discrepancy:
Low Endogenous Expression
The target protein may be expressed at very low levels in tissue samples, below the detection threshold. Increasing the total protein load or enhancing assay sensitivity may help improve detectability.
Inefficient Tissue Extraction
Suboptimal lysis conditions may lead to incomplete solubilization of tissue proteins, including the target protein. Optimizing the lysis buffer composition and extraction protocol is recommended.
Antibody Specificity Limitations
In-house antibodies may demonstrate high affinity for recombinant protein epitopes but reduced recognition of native epitopes in tissue due to conformational differences or interference by other proteins. Further purification or affinity maturation may enhance specificity.
Suboptimal Antibody Concentration
The antibody dilution used for recombinant protein detection may not be suitable for tissue samples. Empirical optimization of primary and secondary antibody concentrations may be required.
Proteolytic Degradation in Tissue Samples
Target proteins may be degraded during sample handling. Immediate homogenization in the presence of protease inhibitors and rapid processing are essential to minimize degradation.
Technical Variability
Inconsistencies in experimental execution may affect reproducibility. Maintaining standardized protocols across experiments is critical for reliable comparative analysis.
Addressing this issue involves a comprehensive optimization strategy, including improvements in tissue extraction efficiency, antibody specificity and concentration, and detection sensitivity, while ensuring consistency in experimental handling and processing.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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