Small wells in western blot

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Paul Rutland

Are you rinsing the wells out just before loading? Sometimes acrylamide leaks out of the gel and forms a dense layer in the well and your sample may sit on this layer not on the bottom of the well.Just a few squirts of running buffer in each well will clean the wells

Aarya Chitransh

If you're experiencing small wells in your polyacrylamide gel for Western blotting, there are several potential reasons for this issue:

Insufficient polymerization: Ensure that the polyacrylamide gel has fully polymerized before loading your samples. Incomplete polymerization can lead to small or collapsed wells. Make sure to follow the manufacturer's instructions for polymerization time and conditions, and consider increasing the polymerization time if necessary.

Improper gel casting: Pay attention to the gel casting process to ensure that the gel is evenly poured and that there are no air bubbles trapped in the wells. Air bubbles can cause irregularities in the gel structure and result in small wells.

Degraded acrylamide: Check the quality of your acrylamide solution to ensure that it is not degraded. Degraded acrylamide can lead to poor gel formation and small wells. Freshly prepare acrylamide solutions and store them properly according to manufacturer's recommendations.

Incorrect buffer composition: Verify that the buffer used for gel electrophoresis is prepared correctly and has the appropriate concentration of ions and additives. Deviations from the recommended buffer composition can affect gel formation and well size.

Sample loading volume: Make sure that you are loading a sufficient volume of sample into the wells. If the volume of sample loaded is too small, the wells may appear smaller than expected. Consider increasing the volume of sample loaded into the wells.

Gel drying: If you are drying the gel prior to Western blotting, ensure that the gel is dried evenly and completely. Uneven drying can cause shrinkage of the gel, resulting in smaller wells.

Equipment issues: Check the condition of your gel casting apparatus and electrophoresis setup for any mechanical issues that may affect gel formation. Make sure that the gel casting plates are properly aligned and that there are no leaks or other problems.

By carefully troubleshooting these potential issues, you should be able to identify and resolve the cause of small wells in your polyacrylamide gel for Western blotting.

Ru Nm

thank you , I will try what you suggested.

Shubhanshu Yadav

You can clear the wells by rinsing it a few times with running buffer it should help