Unexpected or multiple bands in western blot

Protein degradation

Proteases may have digested the protein.

This is likely if you see multiple bands at low molecular weight, or smearing below the expected band.

• Add protease inhibitors to prevent protein degradation.

• Use fresh lysate.

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.

• Try boiling the sample for longer in Laemmli buffer during sample preparation.

Post-transcriptional modifications, including glycosylation.

This is likely if you see multiple bands at high molecular weight, including a smear or streak above the expected band.

• Use un-boiled lysate.

• Check the literature to see where the cleavage site is and which fragment contains the immunogen sequence of your antibody.

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.

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

• Use knock-out lysates or cells that do not express endogenous target protein.

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.

Insoluble proteins are "stuck" in the well.

Insoluble proteins above separation range (>250 kDa) may give non-specific signal at the top/bottom of the gel if "stuck" in the well. Alternatively, dye front that isn't excised before transfer can also yield non-specific signal.

• Remove the stacking layer and dye front before transferring.
• Check protein database/literature for possible sizes of the protein of interest.