Western Blot Troubleshooting and Tips




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The technique of Western blotting aims to identify specific proteins within a complex mixture. Initially, the sample(s) must be resolved (based on size) through Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE), followed by transfer and immobilization on to a membrane (PVDF or nitrocellulose) prior to antibody based detection.

Before running a Western blot, it is extremely important to research the target protein thoroughly. As the Western blot process is sequential, care should be taken at each step to increase the chances of successful detection. The detection steps of a Western blot can vary considerably and will also depend on whether the method used is direct or indirect detection.

The more common Western blot problems seen during this process are detailed below, along with their probable cause and potential solution:

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Weak or No Signal

Primary and / or secondary antibody issues• Optimize the primary and / or secondary antibody concentration(s)
• Replace secondary antibody with Lightning-Link antibody conjugation (for labeling primary antibodies, proteins or peptides)
• Optimize incubation time and / or temperature
Very low quantities of target protein • Check the protein loading and / or increase the amount of protein in each well
• Consider the following upstream activities:
- Low abundance protein enrichment by immunoprecipitation, fractionation etc.
- Treat appropriately to induce target protein expression or modification
- Ensure protein sample has not degraded and / or include protease inhibitors in lysis buffer
- Ensure use of the optimum lysis buffer for the target protein subcellular localization
Transfer / loss of protein through the membrane• Decease the membrane pore size* and / or transfer time (*Expedeon’s PVDF and nitrocellulose membranes are 0.2µM)
• Remove residual SDS by washing the gel in water
Not enough protein transferred to the membrane • Use PVDF membrane
• Soak the gel in 0.05% SDS solution for 5 minutes prior to transfer
• Increase voltage and transfer time
Incorrect membrane type• Choose either PVDF or nitrocellulose membranes based on the hydrophobicity / hydrophilicity of the target antigen as described in the literature:
- PVDF: more suited to hydrophilic / polar / charged target antigens
- Nitrocellulose: more suited to hydrophobic / nonpolar target antigens
Problems with blocking buffer• Reduce the percentage of the blocking reagent from the antibody incubation buffers
• Use a different blocking reagent
Low molecular weight (MW) target proteins• Reduce transfer times for low MW proteins <30kDa
Unsuccessful / incomplete transfer of protein• Insufficient / no current during the transfer run
• Incorrect transfer buffer formulation / dilution
• Protein should be incubated with SDS and used with a PVDF membrane
• Ensure that the transfer stack and procedure has been set up correctly (e.g., no air bubbles trapped between the gel and the membrane)
• A thicker gel during electrophoresis (especially if using a handcast gel) can lead to incomplete transfer of high MW proteins
• The quality of protein transfer can be checked by using a reversible, universal protein stain, e.g., Ponceau S
• Compared with semi dry transfer, wet transfer produces higher resolution
SDS concentration too high / low• SDS coats the proteins and allows them to transfer – Increase SDS for the transfer of large MW proteins and remove SDS for small MW protein transfer
Contamination with sodium azide • Use sodium azide free buffers when using HRP
• Undertake thorough washing step(s)
Incorrect pH of transfer buffer • Check the pH of transfer buffer to ensure that it is correct
Transfer time too short• Increase transfer time
Incorrect assembly and orientation of transfer stack• Ensure correct assembly and orientation of the transfer stack
Detection reagent not sensitive enough• Try a different detection reagent (e.g. Lightning-Link antibody conjugation (for labeling primary antibodies, proteins or peptides)
• Try a different detection reagent
Chemiluminescent reagents should be diluted in high purity water


High Background and Nonspecific Bands

Antibody is too concentrated• Titrate / dilute antibody
Membrane not blocked correctly• Increase the concentration of blocking reagent
• Increase blocking time and / or temperature
• Addition of Tween 20 to the blocking buffer, or consider adding both to the primary antibody dilution buffer
Not enough washing or wash steps• Increase washing time and volume
Membrane has dried out• Prevent membrane from drying out during immunoblotting
Nonspecific binding of secondary antibody• Remove excess secondary antibody for reduced cross reactivity
• Replace secondary antibody with Lightning-Link antibody conjugation (for labeling primary antibodies, proteins or peptides)
• For phosphorylated protein detection, milk based buffers should not be used (milk and casein are rich in phosphoprotein)

Detection reagent is too sensitive• Investigate other types and / or dilution of the detection reagent
The amount of target protein is lower than the threshold of nonspecific binding• Low abundance protein enrichment by immunoprecipitation, fractionation, etc
Target sample has degraded• Greater potential for nonspecific bands and degradation products from tissue extracts
• Use freshly prepared lysates and include protease inhibitors (and phosphatase inhibitors for the detection of phosphorylated targets)
Presence of isoforms and / or post translational modifications (PTMs)• Conduct a literature search of your protein to establish the presence of isoforms / PTMs
• PTMs (glycosylation, phosphorylation, precursor maturation, etc.), can lead to a change in band sizes
• Use an antibody that is specific for the isoform


Other Western Blot Problems

Hollow ‘ghost’ bands• These are seen when an ECL substrate is converted too rapidly
White bands on a dark blot - known as inverse staining• This is due to excess primary and / or secondary antibody
• Replace secondary antibody with Lightning-Link antibody conjugation (for labeling primary antibodies, proteins or peptides)
• Increase the dilutions of your antibodies
Staining of the MW marker• The antibody has reacted with the MW marker
Protein bands appear uneven• Protein concentrations that are too high or loaded unevenly can result in diffuse protein bands
• Gel composition is not uniform (gel has set too quickly while casting, or the gel buffer was mixed inadequately)
• Uneven bands can be due to not enough buffer being added to the tank during running
White spots / blank spaces• Can be caused by improper / uneven transfer or from air bubbles
• Use a roller to remove air bubbles
• Under or over compression of the transfer stack – check transfer stack layers and sponges
Dark dots / spots• Binding of antibodies to the blocking reagent in the blocking buffer – Replace and / or filter the blocking reagent
• Remove excess detection reagent from the membrane by washing
Biotin reagents• No signal is produced when milk based blockers are used with biotin reagents


  • Bass, JJ., Wilkinson, DJ., Rankin, D., Phillips, BE., Szewczyk, N. J., Smith, K., & Atherton, P. J. (2016). An Overview of Technical Considerations for Western blotting Applications to Physiological Research. Scandinavian Journal of Medicine & Science in Sports. 2016;27(1):4–25. doi:10.1111/sms.12702.
  • Gingrich, JC., Davis, DR., & Nguyen, Q. Multiplex Detection and Quantitation of Proteins on Western Blots Using Fluorescent Probes. BioTechniques. 2000;29(3):636–642. doi:10.2144/00293pf02.