Introduction
Gel electrophoresis is a fundamental technique in molecular biology, widely used for the separation of proteins, nucleic acids, and other macromolecules. Proper control of sample volume, voltage, and electrophoresis time is crucial for achieving accurate and reproducible results. Our lab colleague offers best practices for managing these parameters during SDS-PAGE gel electrophoresis.
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Sample Volume: Ensuring Consistency
When performing SDS-PAGE electrophoresis, the sample volume is a key factor that can significantly impact the resolution of your results. It is generally recommended to load 10 µL of total protein per well. To ensure consistency and prevent sample diffusion between adjacent wells, it is important to load an equal volume of 1x loading buffer in any empty wells. This precaution helps to prevent the spreading of samples into neighboring lanes, which can occur if the well is left empty.
Before loading your samples, always start by adding a molecular weight marker to one well. This allows for easy identification of protein sizes after electrophoresis.
Voltage Control: Balancing Speed and Resolution
The voltage applied during electrophoresis directly affects both the speed at which samples migrate through the gel and the resolution of the separation. For SDS-PAGE, it is advisable to start with a low voltage of around 80V. This initial low voltage allows the samples to migrate slowly and evenly, concentrating them in a sharp band as they enter the separating gel.
Once the samples have fully entered the separating gel, the voltage can be increased to 120V. This higher voltage accelerates the migration, ensuring that the proteins are efficiently separated according to their molecular weight. It is essential to monitor the progress of the bromophenol blue dye front, which indicates the completion of the electrophoresis. For gels with a concentration of 10-12%, 80-90 minutes is typically sufficient; however, for 15% gels, you may need to extend the run time slightly.
Time Management: Knowing When to Stop
Timing is another critical factor in gel electrophoresis. Running the gel for too long or too short a period can lead to suboptimal separation. The migration of the bromophenol blue dye is a useful indicator: when it reaches the bottom of the gel, it is usually time to stop the run. For standard gels, such as 10-12%, an electrophoresis duration of about 80-90 minutes is typically adequate. For higher percentage gels, like 15%, the run time should be extended to ensure complete separation of proteins.
Buffer Management: Reusing and Preparing Buffers
Electrophoresis buffer can be reused 1-2 times, depending on your laboratory’s specific conditions. However, for optimal results, it is recommended to prepare fresh 10x buffer and dilute it just before use. This ensures that the buffer maintains its effectiveness, leading to more reliable electrophoresis results.
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By carefully controlling the sample volume, voltage, and electrophoresis time, you can significantly improve the accuracy and reproducibility of your gel electrophoresis results. Implementing these best practices in your laboratory work will help you achieve clearer and more distinct bands, leading to better data for downstream analysis.
If you have more good methods to optimize gel electrophoresis experiment, welcome to discuss with us!
Beijing Liuyi Biotechnology Co. Ltd (Liuyi Biotechnology) has specialized in manufacturing electrophoresis instruments for more than 50 years with our own professional technical team and R&D center. We have reliable and complete production line from design to inspection, and warehouse, as well as marketing support. Our main products are Electrophoresis Cell (tank/chamber), Electrophoresis Power Supply, Blue LED Transilluminator, UV Transilluminator, Gel Image & Analysis System etc. We also supply the lab instruments like PCR instrument, vortex mixer and centrifuge for laboratory.
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Post time: Aug-20-2024