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Western Blot Troubleshooting: Weak/No Signal & Other

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Overcome your Western blot difficulties with our troubleshooting advice, covering problems such as weak/no signal, non-specific bands, high signal, and other common issues.

Issues with the primary and / or secondary antibody

  • Titrate the antibody to determine optimum concentration. 

  • The antibody may have lost activity – perform a dot blot to determine activity and optimal concentration. 

  • Include a positive control (e.g., overexpressed protein, purified protein, positive cell line, etc. Adjust protein loading accordingly). 

  • Change incubation time and temperature (4°C, overnight).

Target protein abundance is too low

  • Load more protein per well. 

  • Enrich low-abundance proteins by immunoprecipitation, fractionation, etc. 

  • Use appropriate treatment to induce target protein expression or modification. 

  • Ensure sample has not degraded. 

  • Include protease inhibitors in the lysis buffer. 

  • Use the optimum lysis buffer for the target protein’s subcellular localization. 

  • Check protein loading with an internal loading control antibody.

Membrane choice

Select PVDF or NC membranes based on hydrophobicity/ hydrophilicity of the target antigen. 

Blocking buffer issues

  • Blocking for too long can mask specific epitopes and prevent antibody binding. 

  • Reduce the percentage of, or remove, the blocking reagent from the antibody incubation buffers. 

  • Switch to using an alternative blocking reagent.

Low molecular weight targets

  • Use a Tris-tricine gel for protein targets <20kDa.

  • Reduce transfer times and/or use smaller pore size membranes (0.22 μm) for low MW proteins <30kDa.

  • Wet transfer is recommended for small proteins (10kDa).

Unsuccessful transfer

  • Ensure proper transfer set-up (e.g., no air bubbles trapped between the gel and the membrane). 

  • Thicker gels can result in incomplete transfer of high molecular-weight-proteins. 

  • Check the quality of protein transfer with a reversible, universal protein stain, e.g., Ponceau-S. 

  • Wet transfer produces higher-resolution transfers over semidry transfer.

Sodium azide contamination

  • The presence of sodium azide inhibits the activity of HRP. 

  • Use sodium azide-free buffers. 

  • Ensure sufficient washing.

Film exposure too short / detection reagent not sensitive enough 

  • Check several exposure times to achieve optimum detection. 

  • Try different detection reagent compositions and/or brands. 

  • Dilute chemiluminescent reagents in high-purity water.

 
Other blotting issues
Ghost hollow bands
  • This happens when the ECL substrate is used up too rapidly.
Sample overloading 
  • Decrease the total protein loading for each sample.
Too much antibody 
  • Decrease the concentrations of the primary and/or secondary antibodies.
Inverse staining (i.e., white bands on a dark blot) 

  • Too much primary and/or too much secondary antibody.

  • Use antibodies at higher dilutions.

Molecular weight marker staining 

  • The antibody reacts with the MW marker. 

  • Add a blank lane between the MW marker and the first sample lane.
“Smiling” bands 

  • Migration through the gel was too hot or too fast. 

  • Reduce the voltage applied to run the SDS-PAGE gel or run the gel in a cold room.

Blank areas/white spots
  • Can be caused by improper/uneven transfer or air bubbles.
Uneven bands 

  • High protein concentrations can result in diffuse protein bands. 

  • Uneven protein loading: assay protein samples and load by protein amount. Check for even protein loading by stripping and reprobing the blot with an internal control antibody (or use an HRP-conjugated loading control antibody). 

  • Uneven gel composition (gel has set too quickly while casting or buffer was mixed inadequately). 

  • Uneven bands can be due to insufficient buffer being added to the tank during running.

Dark spots/dots 

  • This problem can be caused by antibodies binding to the blocking reagent in the blocking buffer. 

  • Change to another blocking reagent. 

  • Filter the blocking buffer. 

  • Wash excess detection reagent from the membrane before exposure.