Protein electrophoresis is a powerful technique used in many laboratories for the separation and analysis of proteins. The electrophoresis chamber is a crucial component of this process, and proper usage is essential for obtaining reliable results. Here are some best practices and tips for using a protein electrophoresis chamber effectively.
1. Preparation and Setup
Buffer Preparation
- Fresh Buffers: Always prepare fresh running and sample buffers. Old or contaminated buffers can lead to inconsistent results. If you can't make or use fresh buffer, keep track of how many times used buffer has been run.
- pH Levels: Ensure the pH of your buffers is correct, as this can significantly impact protein migration.
Gel Preparation
- Casting Gels: Use clean, grease-free plates and ensure the gel is polymerized completely before use. Don't forget to remove the stickers on the sides or bottom of the gel
- Gel Storage: If preparing gels in advance, store them in an appropriate buffer at 4°C to prevent drying out, preferably on a rocker.
2. Sample Preparation
Sample Quality
- Protein Concentration: Ensure your protein samples are at the appropriate concentration for detection.
- Reducing Agents and Denaturants: Use reducing agents (e.g., DTT, β-mercaptoethanol) and denaturants (e.g., SDS) as needed to ensure proteins are in their denatured state, providing consistent results. Boil samples before running, re-boil each time if re-running samples.
Loading Samples
- Loading Dye: Mix your samples with the appropriate loading dye to add density and visualize the progress.
- Avoid Overloading: Do not overload wells, as this can cause smearing, spillover, and poor resolution.
3. Running the Gel
Setting Up the Chamber
- Gel Orientation: Ensure the gel is correctly positioned in the chamber, with the wells facing the correct direction.
- Buffer Levels: Fill the chamber with the running buffer, making sure both the upper and lower reservoirs are adequately filled.
- Track Wells: Write down which sample is going into which well before you load them.
Voltage and Time
- Consistent Voltage: Set the voltage according to the protocol. Running at too high a voltage can cause overheating and poor resolution. Some scientists run at a low voltage for the first 5-10 minutes then crank up the voltage to speed it up.
- Monitoring: Regularly monitor the progress of the electrophoresis to avoid running the dye front off the gel.
4. Post-Run Procedures
Staining and Destaining
- Staining: Use the appropriate staining method (e.g., Coomassie Brilliant Blue, silver stain) to visualize proteins. There are Instant staining and destaining options available avoid the hours Coomassie requires, the instant ones take 10-20 minutes total.
- Destaining: Destain the gel adequately to reduce background staining and improve band visibility.
Documentation
- Imaging: Use a gel documentation system to capture images of your results. Ensure the settings are optimized for the best contrast and clarity.
- Analysis: Use software for quantitative analysis of band intensity if required.
5. Maintenance and Care of the Electrophoresis Chamber
Cleaning
- After Each Use: Rinse the chamber and electrodes with distilled water after each use to prevent buffer salt buildup.
- Periodic Deep Cleaning: Perform a deep clean periodically using mild detergent to remove any stubborn residues.
Inspection
- Check for Damage: Regularly inspect the chamber, electrodes, and power leads for any signs of wear or damage.
- Replace Worn Parts: Replace any damaged or worn parts immediately to avoid compromising your results.
6. Safety Considerations
Electrical Safety
- Handle with Care: Always handle the electrophoresis chamber and power supply with dry hands and in a dry environment.
- Turn Off Before Adjusting: Always turn off the power supply before making any adjustments to the gel or chamber.
Chemical Safety
- PPE: Wear appropriate personal protective equipment (gloves, lab coat, safety glasses) when preparing and handling buffers and gels.
- Waste Disposal: Dispose of chemical waste according to your institution's safety guidelines.
Conclusion
Using a protein electrophoresis chamber correctly is essential for obtaining high-quality, reproducible results in your protein analysis experiments. By following these best practices and tips, you can ensure that your electrophoresis runs smoothly and efficiently, leading to clearer, more interpretable results. Proper maintenance and safety precautions will also extend the lifespan of your equipment and protect you from potential hazards.