Western Blotting Troubleshooting – CYANAGEN TIPS – Non-Specific Bands

Sometimes, at the end of a Western blot, non-specific bands appear on the membrane.
Several factors may cause these non-specific bands.
We provide hints to fix the issue for each root cause.

• Aggregation of analyte. Increase the reducing agent to ensure a complete reduction of disulfide bonds.

• SDS interference. The presence of SDS may result in the development of unspecific bands caused by antibodies binding to the charged SDS molecules associated with the proteins. Wash the membrane thoroughly after transfer with water.

• High protein concentration. A commonly seen effect is the diffusion of protein bands. Reduce the amount of protein initially loaded.

• The primary antibody is not specific to the protein of interest. Use monospecific or antigen-affinity purified antibodies. Incubate your primary antibody continuously at 4°C overnight, not at room temperature. Reduce NaCl in TBS-T Buffer (100mM÷350mM). Use monospecific or antigen-affinity-purified Ab.

• Non-specific binding of secondary antibody. Confirm the secondary is specific by omitting the primary and running a secondary-only blot. If bands develop, choose an alternative secondary antibody.

Cyanagen developed three ECL substrate lines:

WESTAR (ECL two-solution format), more details at http://bit.ly/4t5XREH
WESTAR-ONE (ECL one-solution format), more details at https://bit.ly/3MdndzS
WESTAR-TAB (ECL tablet format), more details at https://bit.ly/4qJy1ER

REQUESTS for samples: sales@cyanagen.com