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Bands are misshapen or uneven in Western blot

Explore possible causes and solutions for misshapen or uneven bands in Western blot.

Smile effect on bands

Possible causes

Solution

Voltage may have been too high during migration.

If the voltage is too high, migration will occur too quickly.

  • Check our western blot protocol for the suggested voltage and decrease if necessary.

Gel may have been too hot during migration.

If the temperature is too high, the pH of the buffer may be slightly altered, which could affect migration.

  • Run the gel at 4°C: on ice or in a cold room.

Bands are uneven

Possible causes

Solution

The gel has polymerized unevenly.

When the gel has not polymerized properly, bands can appear wonky or uneven. In extreme cases, lanes probed for the same protein can appear at different molecular weights.

  • Check your gel recipe to see if you've added the right amount of TEMED.

  • Ensure the gel is covered entirely in buffer while it is setting.