Western blotting is a powerful technique, but it’s also susceptible to errors that can compromise your results. Automating as many steps as possible will reduce the chance of error and improve overall results. The following are suggestion that can reduce your chance of error:
Sample preparation:
- Protease inhibitors: Include protease inhibitors in your lysis buffer to prevent protein degradation before and during sample preparation.
- Protein quantification: Accurately quantify your protein samples to ensure equal loading on the gel. This is crucial for comparing protein expression levels between samples.
- Sample storage: Store protein samples properly at -80°C to minimize protein degradation and maintain sample integrity.
Gel electrophoresis:
- Gel quality: Use high-quality gels with the appropriate percentage and pore size for your proteins of interest. Leaky or uneven gels can lead to poor separation and band distortion.
- Loading: Load samples carefully to avoid well distortion and ensure even protein distribution in each lane.
- Running conditions: Maintain consistent running conditions (voltage, amperage, time) to ensure reproducible separation of proteins.
Transfer:
- Transfer buffer: Use fresh transfer buffer with the appropriate composition for your chosen transfer method (wet, semi-dry, etc.).
- Membrane activation: Activate PVDF membranes properly before transfer to optimize protein binding.
- Transfer efficiency: Monitor transfer efficiency using Ponceau S staining to ensure complete transfer of proteins from the gel to the membrane.
Blocking and antibody incubations:
- BlotCycler Touch or Mini: Both instruments are ideal at this stage of the blot processing cycle. The automated process eliminates the variations in fluid delivery leading to consistent reproducible results.
- Blocking buffer: Use an appropriate blocking buffer to prevent non-specific antibody binding. Common choices include milk powder, BSA, or casein in TBST.
- Antibody concentrations: Optimize antibody concentrations to achieve specific binding with minimal background noise. Start with recommended dilutions and adjust as needed.
- Incubation times and temperatures: Follow recommended incubation times and temperatures for each antibody to ensure optimal binding specificity.
Detection:
- Substrate choice: Choose the appropriate detection substrate (HRP, AP) based on your secondary antibody and desired signal intensity.
- Washing steps: Thoroughly wash the membrane between incubation steps to remove unbound antibodies and reduce background noise. All washing steps are automated using the BlotCycler.
- Exposure time: Adjust exposure time for chemiluminescent detection to achieve optimal signal without overexposure.
Additional Suggestions:
- Use fresh reagents: Expired or degraded reagents can significantly affect blotting results.
- Maintain a clean working environment: Minimize dust and contamination in your workspace to prevent protein degradation and non-specific binding.
- Keep detailed records: Document all steps of your protocol, including reagents, incubation times, and temperatures, for easier troubleshooting and reproducibility.
- Positive and negative controls: Include positive and negative controls in your blots to validate the specificity of your antibodies and rule out non-specific binding.
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