How to Solve Undetectable Target Protein in Eukaryotic Overexpression WB?
When overexpressing a target protein in a eukaryotic system but failing to detect it via Western Blot (WB), several factors may be responsible. Here are some diagnostic steps and strategies to address this issue:
Issues with Detection Method
1. Ensure correct WB procedures, including sample preparation, electrophoresis, transfer, and antibody probing.
2. Check protein sample concentration; low levels may prevent detection.
3. Verify electrophoresis and transfer efficiency to confirm protein transfer to the membrane.
4. Optimize antibody selection and usage, adjusting concentration or incubation time if necessary.
Target Protein Expression Issues
1. Assess protein expression levels; optimize transfection conditions, expression time, or use a more efficient system.
2. Evaluate protein stability; if degradation or rapid transport occurs, use stabilizers or inhibitors.
3. Consider subcellular localization; adjust lysis methods for nuclear or cytoplasmic extraction.
Antibody Issues
1. Confirm antibody specificity and affinity for the target protein.
2. Try different antibodies from various sources or clones.
3. Validate results with multiple antibodies targeting different protein regions.
Transfection/Transformation Efficiency
1. In mammalian expression systems, ensure high transfection efficiency.
2. In yeast or microbial systems, use appropriate transformation methods and host strains.
Protein Modification and Localization
1. Post-translational modifications may alter migration rates, affecting apparent molecular weight.
2. If the protein localizes to specific compartments (e.g., nucleus), ensure effective extraction.
Other Considerations
1. Check experimental conditions, such as temperature and pH.
2. Use alternative detection methods like immunohistochemistry or fluorescence staining to confirm expression.
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