Perhaps more than any other step in Western blotting, immunodetection conditions are subject to the greatest number of variables, in part because this step depends on antibody:antigen recognition. Our scientists know how tedious it can be to optimize immunodetection parameters; that’s why we developed the vacuum-driven SNAP i.d.® Protein Detection System, cutting the time for blocking, probing, and washing to 30 minutes.

Go ahead, troubleshoot – and to learn more about fast immunodetection, click here.

Click on the immunodetection topics to read about the possible causes and remedies:

Weak Signal

Possible Cause Remedy

Improper blocking reagent
  • The blocking agent may have an affinity for the protein of interest and thus obscure the protein from detection. Try a different blocking agent and/or reduce both the amount or exposure time of the blocking agent.
  • Explore blocking reagents

Insufficient antibody reaction time
  • Increase the incubation time.

Insufficient signal amplification
  • Switch from a monoclonal to a polyclonal primary antibody. In polyclonal antibodies, the presence of multiple epitopes on the same protein can generate greater signal.
  • If using a conjugated primary antibody, switch to an unconjugated primary antibody and secondary antibody, which will increase the sensitivity of detection.
  • Biotin-conjugated antibodies provide greater sensitivity and higher amplified signal when compared to fluorochrome- or enzyme-conjugated secondary antibodies.
  • Search for antibodies using our Antibody Finder

Antibody concentration is too low or antibody is inactive
  • Multiple freeze-thaw cycles, bacterial contamination, or repeated use of antibody solution can change antibody titer or activity. Increase antibody concentration or prepare it fresh.
  • For fluorescent secondary antibodies, ensure that the antibody stock vial and any aliquots are protected from light.

Antibody not suitable for Western blotting or not compatible with preparation of cells/tissue

Outdated detection reagents

Protein transfer problems
  • Optimize protein transfer (see above).

Dried blot in chromogenic detection
  • If there is poor contrast using a chromogenic detection system, the blot may have dried. Try rewetting the blot in water to maximize the contrast.

Tap water inactivates chromogenic detection reagents

Azide inhibits  HRP
  • Do not use azide in the blotting solutions.

Antigen concentration is too low
  • Load more antigen on the gel prior to the blotting.

No Signal

Possible Cause Remedy
Incompatible primary and/or secondary antibodies Whenever possible, ensure that the primary antibody is raised against the sequence of the protein found in the same species as your sample. For example, use an antibody targeting the human form of your protein if measuring protein levels in human tissue. If an identical sequence is not possible, determine sequence overlap and reactivity at

Secondary antibodies target the animal species that the primary antibody was raised in. Only use a secondary antibody that was raised in a different species than the host species of the primary antibody, and that is as phylogenetically far from the species of your sample as possible. In addition, ensure that the primary and secondary antibody classes are matched. Most primary antibodies belong to the IgG class, and should be used with a secondary antibody specific for IgG.

Antibody concentration too low

HRP inhibition
  • HRP-labeled antibodies should not be used in solutions containing sodium azide.

Primary antibody was raised against native protein
  • Separate proteins in non-denaturing gel or use antibody raised against denatured antigen.

Detection reagent is not sensitive enough
  • Use a more sensitive detection reagent, with a more appropriate range of detection.
  • Learn about Luminata™ detection reagents for chemiluminescent Western blots

    Adjust the sensitivity of a Western blot using Luminata chemiluminescent reagents for Western blotting, which provide a wide dynamic range of detection.

    Adjust the sensitivity of a Western blot using Luminata chemiluminescent reagents for Western blotting, which provide a wide dynamic range of detection.

Uneven Blot

Fingerprints on a Western blot

Fingerprints on a
Western blot caused
by touching
membrane without
gloves or
Possible Cause Remedy

Fingerprints, fold marks or forceps imprints on the blot

Speckled Background

Speckled background on Western blot

Speckled background
on Western blot
caused by insufficient
washes and/or unfiltered blocking solution.
Possible Cause Remedy

Aggregates in the blocking reagent
  • Filter blocking reagent solution through 0.2 µm or 0.45 µm Millex® syringe filter unit.
  • Browse Millex® filters

Aggregates in HRP-conjugated secondary antibody

High Background

Overall high background in a Western blot

Overall high background in a Western blot
Possible Cause Remedy

Low primary antibody specificity

Secondary antibody concentration is too high
  • Increase antibody dilution.

Secondary antibody cross-reactivity
  • Use a secondary antibody that has been pre-adsorbed with serum from the species of your target cells or tissue.

Insufficient washes

Protein-protein interactions
  • Use Tween®-20 (0.05%) in the wash and detection solutions to minimize protein-protein interactions and increase the signal to noise ratio.

Immunodetection on Immobilon®-PSQ transfer membrane
  • Increase the concentration or volume of the blocking agent used to compensate for the greater surface area of the membrane. Persistent background can be reduced by adding up to 0.5M NaCl and up to 0.2% SDS to the wash buffer and extending the wash time to 2 hours.

Poor quality reagents

Cross-reactivity between blocking reagent and antibody
  • Use different blocking agent or use Tween®-20 detergent in the washing buffer.

Film overexposure
  • Shorten exposure time.

Membrane drying during incubation process
  • Use volumes sufficient to cover the membrane during incubation.

Poor quality antibodies

Excess detection reagents
  • Drain blots completely before exposure.

Detection reagent is too sensitive

Persistent Background

Possible Cause Remedy
Nonspecific binding
Adding 0.5 M NaCl and 0.2% SDS to the wash buffer reduced the background considerably on the Immobilon®-PSQ membrane, which has a high protein binding capacity.

High Background (Pertains to rapid immunodetection protocol only)

Possible Cause Remedy

Membrane wets out during rapid immunodetection
  • Reduce the Tween®-20 (<0.04%) detergent in the antibody diluent.
  • Use gentler agitation during incubations.
  • Rinse the blot in Milli-Q® water after electrotransfer to remove any residual SDS carried over from the gel. Be sure to dry the blot completely prior to starting any detection protocol. 

Membrane was wet in methanol prior to the immunodetection
  • Do not pre-wet the membrane.

Membrane wasn’t completely dry prior to the immunodetection
  • Make sure the membrane is completely dry prior to starting the procedure.

Nonspecific Binding

Possible Cause Remedy

Primary antibody concentration too high
  • Increase primary antibody dilution.
Diluting primary antibody increases specificity

Diluting primary antibody increases specificity.

Secondary antibody concentration too high
  • Increase secondary antibody dilution.

Antigen concentration too high
  • Decrease amount of protein loaded on the gel.

Reverse Images on Film (White Bands on Dark Background)

Burnt out bands

Negative (reverse) image (“bleached” or “burnt-out”
bands) on film caused by excess secondary
HRP-conjugated antibody (left). The detection was
improved by increasing secondary antibody dilution
10-fold (right).
Possible Cause Remedy

Too much HRP-conjugated secondary antibody

  • Reduce concentration of secondary HRP-conjugated antibody.

Poor Detection of Small Proteins

Possible Cause Remedy

Small proteins are masked by large blocking molecules such as BSA
  • Consider casein, a low molecular weight blocking agent, such as polyvinylpyrrolidone (PVP), or a protein-free blocking agent
  • Explore blocking reagents
  • Surfactants such as Tween® and Triton® X-100 may have to be minimized.
  • Avoid excessive incubation times with antibody and wash solution.

Presence of Additional Band(s) or Band(s) at the Wrong Size

Possible Cause Remedy

Nonspecific primary antibody
  • Verification of antibody specificity is a crucial part of any Western blot experiment. In addition to consulting the antibody data sheet for evidence of specificity, you should conduct your own verification. Common ways to assess specificity include an absence of band(s) in cells/tissue lacking the protein of interest, expected expression in immunohistochemistry, and presence of similar bands using another primary antibody targeting the same protein.

Protein degradation
  • Ensure adequate protease/phosphatase inhibitor concentration in sample and prepare cell lysate on ice.

Protein glycosylation, phosphorylation/dephosphorylation, ubiquitination, etc.
Protein cleavage
  • A variety of post-translational modifications can change the molecular weight of a protein. Examine the published literature to support this conclusion.

Large Error Bars in Quantitative Western Blotting

Possible Cause Remedy
Variability in blotting conditions between experimental runs
  • Ensure identical Western blotting conditions across experiments. Use the same antibody lots for all runs of an experiment. Add more replicates.