Western Blot Troubleshooting: Weak/No Signal & Other

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. 

• Check the hydrophobicity/hydrophilicity of the antigen sequence. 

• PVDF membrane works better with hydrophilic/ polar/charged target antigens. 

• Nitrocellulose works better with hydrophobic/ non-polar antigens.

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.