Western Blot Doctor™ — Weak Signal Detection



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 the problem of weak or no signal detection.

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: Weak or no signal detection

Back to Top

A blot with weak-signal – Western Blot Doctor - Weak-Signal Detection

Likely Causes and Recommended Solutions Antibody problems: low activity or concentration, low-affinity primary antibody, mismatched primary and secondary antibodies:
  • Increase antibody concentrations
  • Antibody may have lost activity; perform a dot blot to determine activity and optimal concentration
  • Test different primary antibodies and/or primary/secondary pairs
  • Try to include both a positive control and a negative control
Insufficient amount of antigen present
  • Load more protein in each sample on the gel
  • Check sample integrity; make sure proteins are not degraded
The antigen is masked by the blocking buffer
  • Try different blocking reagents (for example, avoid using milk when detecting phosphoproteins)
  • Optimize blocking reagent concentration — recommendation is 3–5% BSA or NFDM
Chemiluminescent substrate not performing well
  • Use longer substrate incubation time
  • To determine if the substrate has lost activity, prepare a small amount of working solution. In a darkroom, add a small amount of HRP conjugate. A blue light should be observed. If no glow is observed, either the substrate or the HRP conjugate has lost activity.
  • Ensure that there is no cross-contamination between the two bottles in the substrate kit. Contamination between the two substrate reagents can result in a decline in activity.
  • Azide is an inhibitor of HRP; do not use azide in the secondary antibody buffer
Membrane being stripped and reprobed
  • Optimize stripping procedure
  • Reprobe only when necessary
  • Avoid repeated reprobing of the same membrane
Protein not transferred
  • Optimize transfer efficiency for target protein
Tips If you observe non-specific bands on the blot, there are a number of likely causes, mostly related to antibody detection and substrate development as noted above. Problems related to initial protein transfer from the gel to the membrane can also cause weak or no signal; see the Protein Transfer Issues page for more details.


Jensen CE (2012). The Basics of Western Blotting. Anatomical Record 295(3) 369–371.

Protein Blotting Guide — Bio-Rad bulletin 2895

Springer Western Blotting Protocol Book


Ask an Expert

Call us at 1-800-4-BIORAD (1-800-424-6723)

Sign Up for Bio-Rad Updates!
Enter your email address below to receive your choice of the latest news, promotions, and more.