Sample runs out after starting PAGE?

08 November 2022 1 6K Report

After starting the electrophoresis my samples often 'fall' trough the pocket of the gel and accumulate on the stacking/resolving gel border. Every ingredient of the gel had been prepared fresh, the gel was allowed to polymerize long enough and was also stored correctly. Had anyone similar problems and knows how to solve this?

Thank you for your advice.

Nikolay Klyashtornyy

Here are some potential causes and troubleshooting steps to address this issue:

Insufficient sample loading volume: Ensure that you are loading an appropriate volume of your samples into the wells. If you are not loading enough volume, the samples may not be retained in the wells and can migrate into the resolving gel. Increase the sample volume to ensure proper loading.

Incorrect well formation: Check the formation of the wells before loading your samples. Make sure that the comb or well-forming accessory is properly inserted into the gel, creating well pockets that are deep and have straight sides. If the wells are not well-formed, it can lead to sample leakage during electrophoresis.

Buffer compatibility: Confirm that the buffer used for sample loading is compatible with the gel and running conditions. Incompatible buffer compositions can affect the stacking and migration of the samples. Use the appropriate buffer recommended for your specific gel system.

Gel concentration and pore size: Ensure that the gel concentration and pore size are appropriate for your samples. If the gel concentration is too low or the pore size is too large, the samples may migrate too quickly, bypassing the wells and accumulating at the gel border. Consider adjusting the gel concentration or using a different gel with smaller pore sizes.

Electrophoresis conditions: Optimize the electrophoresis conditions, including voltage and run time. Running the gel at a lower voltage or for a shorter duration can help prevent the rapid migration of the samples and improve their retention in the wells.

Sample preparation: Ensure that your samples are properly prepared and mixed before loading. Aggregates, precipitates, or inconsistent mixing can contribute to sample falling through the wells. Centrifuge the samples briefly before loading and mix them thoroughly to achieve uniform distribution.

By addressing these potential issues, you should be able to improve the retention of your samples within the wells during gel electrophoresis. It's always a good idea to perform controls and optimize your protocol to achieve the best results.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

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