Wrong Band Size & Bands Smile Effect Troubleshooting in Western Blots

The Western Blot / WB is a important technique in most labs. By using a Western Blot, researchers are able to know valuable information about proteins of interest, such as molecular weight and post-translational modification. However, wrong band size and smile effect are also common issues in the Western Blotting.

This Western Blot troubleshooting guide can assist you solve wrong band size and smile effect issues.

Western Blot possible causes & solutions for wrong band size

Wrong band size in a western blot

Wrong band size in a Western Blot
Possible cause Solution
Post-translational modification, e.g. glycosylation will make apparent molecular weight larger showed from the membrane • Use a positive control, and make a glycosylation analysis with some glycosylase such as UNG.
Poor protein separation efficiency • Check the gel concentration, and make sure of a higher percentage for small proteins and lower percentage for large proteins.
The structure and amino acid charge of your target protein itself -

Western Blot possible cause & solution for bands smile effect

Bands smile effect in a western blot
Bands smile effect in a Western Blot
Possible cause Solution
Protein migration too fast • Slow down the protein migration.
Too hot • Run the gel in cold environment or on ice.