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Unexpected or multiple bands in Western blot

Explore possible causes and solutions for unexpected or multiple bands in Western blot.

Multiple bands at a low molecular weight

Possible causes

Solution

Proteases may have digested the protein.

Proteases like phosphotase and glycosidase can complicate western blots by denaturing the target protein.

  • Add protease inhibitors to prevent protein degradation.

Multiple bands at a high molecular weight

Possible causes

Solution

The protein may form multimers.

This is likely if you see extra bands at high molecular weights that are 2x or 3x the weight of the expected bands. Some proteins will form dimers, trimers, or larger multimers due to disulfide bond formation if the samples are insufficiently reduced.

  • Boil the sample for longer in Laemmli buffer during sample preparation to prevent this.

Multiple bands at various molecular weights

Possible causes

Solution

The protein may have multiple isoforms or post-translational modifications

Many proteins display bands at slightly higher molecular weights than expected due to post-translational modifications (PTMs) such as phosphorylation, glycosylation or alternative splice variants. 

  • Check the literature to see if multiple bands are reported.

  • To confirm the extra bands are due to PTMs, you may need to break down modified proteins by treating samples with suitable reagents. For example, PNGase F can remove glycosylations. The additional bands should then disappear when running another blot.

The cell line may have been passaged too many times.

Cell lines that have been frequently passaged gradually accumulate differences in their protein expression profiles

  • Go back to the original non-passaged cell line and run these samples in parallel.

The bands may be non-specific.

  • Where possible, use blocking peptides to differentiate between specific and non-specific bands. Only specific bands should be blocked (and thus disappear).

The antibodies are not purified.

Some antibody formats are relatively impure and may contain additional proteins.

  • Use antibodies that have been affinity purified if possible.