Western Blot Doctor™ — Nonspecific Bands



The Western Blot Doctor is a self-help guide that enables you to troubleshoot your western blotting problems. In this section, you can find solutions to issues related to nonspecific bands.

Other sections in the Western Blot Doctor:

Problems and Solutions

Click on the thumbnail that is most representative of your own blot to discover the probable causes and find specific solutions to the problem.



Problem: Nonspecific Bands

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A blot with non-specific-bands – Western Blot Doctor

Likely Causes and Recommended Solutions Antibody concentrations are too high
  • Reduce antibody concentrations
Insufficient blocking of nonspecific sites
  • Increase the concentration of blocking reagent, e.g., from 5 to 7%
  • Increase blocking time and/or temperature
  • Add 0.05% Tween 20 to blocking buffer
  • Prepare antibody dilutions in the same blocking buffer with 0.05% Tween 20
SDS causes nonspecific antibody binding to immobilized protein bands
  • Wash blots after transfer.
  • Do not use SDS during immunoassay procedure
Antibody quality
  • Try different antibodies
 Tips If you observe nonspecific bands on the blot, there are a number of likely causes. Trials with reduced antibody concentrations and incubation times and different blocking reagents and conditions may be necessary to resolve this issue. Small amounts of added Tween may help at the blocking and antibody incubation steps.

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