Although, western blotting is a well-documented immunoassay, perfecting and optimizing your protocol can be tough. There are a number of common issues that may arise when developing new protocols for various reagents. Below you will find a comprehensive guide to help you troubleshoot common issues reported when western blotting.


You can also take a look at our recommended western blotting protocol for additional help. Or, contact for more troubleshooting information.



No or Low Signal

High Background

Multiple or Non-Specific Bands

Diffuse Bands

Smile Effect on Bands

Uneven Staining of Gel

Target Band is Extremely High/Low on Blot

Lane with Protein Ladder is Black

Black Dots on the Blot

BioLegend Western Blot Troubleshooting Guide
What's the issue? Possible Causes  Solution
No or low Signal The primary and secondary antibodies are not compatible. Ensure you are using secondary antibody that binds to your primary antibody (i.e. if your primary is rat, be sure you are using an anti rat secondary).
Insufficient primary or secondary antibody has bound to the protein of interest.  Utilize a higher concentration of antibody and or incubate for a longer period (i.e. overnight at 4°C).
There is not enough antigen.  Load a larger amount of protein onto the gel. Use protease inhibitors and run the recommended positive control.
Overuse of primary antibody. Use fresh antibody (the effective concentration is lowered after each use).
Incubation with detection reagent not sufficient.  Increase the blots incubation time with detection reagent.
Detection reagents are not working. Make sure detection reagents are functional by testing with a different primary antibody.
Poor transfer during blotting. Make sure the transfer apparatus is set up correctly. Ensure you are using the correct transfer times. 
Secondary antibody is inhibited by sodium azide. Do not use sodium azide with HRP-Conjugated antibodies.
Excessive membrane washing.  Reduce washing step repetitions or duration.
Target protein ran off the gel. Use a positive control and a molecular weight marker matched to the size range of the target protein. 
The target protein is not found in high concentrations in your sample. Maximize the target's concentration by enriching the sample beforehand.
The primary or secondary antibody binds to the blocking agent. Utilize a mild detergent or switch to a different blocking reagent. 
Poor binding of proteins to membrane.  Use a membrane with the correct binding capacity. Dry PVDF membranes after transfer to promote strong binding. 
The protein in the species tested is not recognized by your primary antibody.  Run a positive control. Check literature or perform a BLAST alignment to see whether your antibody should react with the target protein. 
High Background  Insufficient washing or blocking.  Increase blocking time or consider using an alternate blocking reagent. Increase the number of washes. 
Concentration of the primary antibody is too high.  Determine optimal antibody concentration through titration. Use a more dilute antibody with longer incubation times (slow targeted binding is best). 
Secondary antibody binding non-specifically, or binding with blocking agent.  Run a secondary control with no primary antibody. 
Overloaded protein. Decrease the amount of protein loaded on the gel, or dilute the sample.
Contamination of equipment or reagents.  Replace reagents, ensure all equipment is properly cleaned. 
Membrane causing high background. PVDF membranes are considered to give higher background than nitrocellulose membranes. 
Membrane dried out during incubation. Ensure membrane is not drying out during the incubation period. 
Incubation temp too high. Incubate membrane at 4°C.
Cross-reactivity of phospho-specific antibodies with blocking agent. The user may have to try different blocking buffers, such as milk, BSA, etc. to reduce non-specific binding while maintaining specific signals. For optimal results, follow the blocking buffer recommendations from your antibody provider.
Multiple or non-specific bands Primary antibody concentration too high. Decrease the concentration of primary antibody. Run secondary control without the primary antibody. 
Excess protein on gel.  Reduce the amount of protein loaded. 
Issues with blocking.  Optimize blocking time and blocking reagent. 
Insufficient washing.  Increase number of wash steps. 
Antibody not properly purified. Use antibodies purified by the affinity method. 
Target protein has been degraded. Use fresh sample. Include protease inhibitors in your sample buffer. 
Frequently passaged cell lines accumulate differences in protein expression profiles.  Retrieve and expand original cell line, run samples in parallel. 
Target protein has several modified forms (acetylation, methylation, glycosylation etc.). Refer to literature, use agent to remove modifications when possible/necessary. 
Protein subtypes have different molecular weights.  Use bioinformatics analysis and review literature to estimate the correct protein size. 
There are splice variants from the same protein family that share similar epitopes.  Check literature and/or perform a blast search to confirm. 
Multimer formation of target protein. Prior to SDS page, boil protein for 10 min to disrupt multimers. 
Diffuse Bands Concentration of antibody too high. Reduce antibody concentration.
Protein transfer too rapid or gel became over-heated during electrophoresis.  Increase the transfer time and/or run gel at 4°C
Too much protein loaded on gel. Decrease the quantity of protein loaded on gel. 
Smile effect on bands Migration was too rapid. Decrease voltage when running gel. 
Temperature during migration was too high. Run gel at 4°C.
Uneven staining of gel Bacterial contamination of antibodies.  Store antibodies as recommended by your antibody provider. Use fresh buffers. 
Insufficient antibody volume.  Ensure that the membrane is completely covered with antibody and incubate with agitation. 
Target band is extremely high/low on blot Separation during electrophoresis not efficient.  Change gel percentage: use a lower percentage for large proteins, a higher percentage for small proteins. 
Lane with protein ladder is black The antibody is reacting with the protein ladder.  Load gel so there is a blank lane between the ladder and the first sample lane. 
Uneven white spots on blot Air bubbles were trapped between the gel and membrane during transfer. Ensure air bubbles are removed when preparing for transfer.
Black dots on the blot  Antibodies are binding to blocking agent.  Filter blocking agent.