Trouble Shooting

Multiple and/or unspecifc bands

Excess protein loading or detection system too sensitive

Antibody specificity is the property of an immunoglobulin that enables it to bind exclusively (or with markedly higher affinity) to a particular antigen while exhibiting minimal or no binding to other, structurally unrelated epitopes.
Even the best antibody will have weak cross-reactivity to off-targets. Increasing the protein amount loaded to the gel will lead to high protein concentrations on the membrane that can create a dense adsorptive surface that promotes nonspecific IgG binding. Increasing the primary antibody concentration even increases this undesired effect. Similarly, highly sensitive detection methods, such as enhanced chemiluminescence, can reveal nonspecific interactions.

Performing a dilution series of the sample can help distinguish true signals from artifacts.

Low antigen abundance

SDS-PAGE can resolve only about 50–100 distinct bands. If the target antigen represents less than ~0.2% of total protein, detection may be challenging. For example, Synaptobrevin/VAMP co-migrates with histones in crude homogenates, making it difficult to visualize. Attempts to enhance signal intensity may inadvertently produce artificial bands.

Antigen enrichment, through fractionation or immunoprecipitation, should be considered.

Inefficient Transfer

Sometime incomplete or uneven transfer of proteins to the blotting membrane is observed.

Air bubbles trapped between gel and membrane can lead to faint or missing bands in the area of the bubble because they block the current and prevent protein migration.
Wet the membrane with transfer buffer before blot assembly and use a roller or pipette to gently push out bubbles.
In wet transfer (tank blot) incompletely submerged blot sandwiches lead to dry areas with high resistance. Make sure the the blot sandwich is completely wet. Use a tray deep enough to keep the sandwich fully immersed.
A misaligned transfer stack may also lead to suboptimal protein transfer. Align the stack using a ruler or a pre‑cut mask; keep all pieces the same size.
Old degraded buffer may lead to poor transfer. Prepare fresh buffer
Wrong buffer composition. Make sure that the transfer buffer has been prepared according to the manufacturer’s instructions of the blotting device.
Wrong transfer conditions (time, voltage and current settings) can lead to over- or under-transfer of proteins. Follow the manufacturer’s instructions of the blotting device.

Inefficient blocking

A variety of blocking agents are described in the literature, including detergents and protein-based blockers. Inadequate or suboptimal blocking may increase nonspecific binding.

Adjusting the blocking reagent or blocking duration can often resolve these issues.

Proteolytic degradation of the antigen

Proteolysis can occur, especially if samples are not freshly prepared, are stored for extended periods, or are subjected to fractionation after homogenization. Proteolytic fragments typically appear as additional bands of lower molecular weight than the full-length protein.

Keep your samples cooled at every preparation step.
Avoid contaminations.
Including protease inhibitors such as PMSF, pepstatin, leupeptin, or commercially available cocktails is strongly recommended.
Including EDTA sequesters metal ions that are essential for the function of metalloproteases.

Alternative splicing

Several bands are not always the result of suboptimal experimental conditions or cross reactivity. Many proteins have several isoforms, and an antibody may detect more than one of them.
Alternative splicing produces mRNA isoforms that encode protein isoforms that often differ in amino‑acid length, domain composition or post‑translational modification (PTM) sites. When the protein mixture is run on an SDS‑PAGE each isoform can migrate to a different apparent molecular weight (MW). Probing with an antibody directed against a shared epitope can give rise to multiple bands.

Figure 1: Detection of Synaptotagmin7 in WT and KO mouse brain lysate with rabbit anti Synaptotagmin7 antibody (cat. no. 105 173, dilution 1:1000).

Several splice variants are detected by this antibody, that disappear in the KO control proving the specificity of the signals detected.
The low molecular weight band present in all three samples has to be considered as unspecific.

Figure 1: Detection of Synaptotagmin7 in WT and KO mouse brain lysate with rabbit anti Synaptotagmin7 antibody (cat. no. 105 173, dilution 1:1000).

Burned (oversaturated) bands (ECL detection)

In enhanced‑chemiluminescence (ECL) detection, horseradish peroxidase (HRP) catalyzes a rapid luminescent reaction as soon as the substrate cocktail is added to the membrane. If the target protein is abundant and the primary‑/secondary‑antibody pair binds with high affinity, the reaction can generate a burst of light that exhausts the substrate before the camera or imaging system is even triggered. The resulting image contains “burned” or oversaturated bands that obscure quantitative information.

Prepare a dilution series of both primary and secondary antibodies (e.g., 1:2,000; 1:5,000; 1:10,000).
Add the ECL substrate and immediately start imaging. Start with very short (1–2 s) exposure time.

By simultaneously lowering the concentrations of both primary and secondary antibodies, and by minimizing the time between substrate addition and image capture, the flash of light is spread over a longer period, preventing burned bands while preserving sensitivity.

General fluorescent background

General fluorescent background can be due to Coomassie or bromphenole blue (BPB) contamination on the membrane.

Do not run the blue loading dye running-front out of the gel but stop gel before; then cut it off the gel before transfer. If that is not possible please use Orange G as loading dye.
For Wet Transfer (Tank Blot) clean tank with methanol to remove all remains from gel runs; BPB front dye did accumulate in your tanks for years and will end up on the membrane leading to high background.
Use fresh transfer pads/sponges or Whatman paper only.
Use clean incubation boxes. Traces of Coomassie or BPB can cause high background.
PVDF membranes show autofluorescence.
Use Nitrocellulose or low fluorescent PVDF membranes. PVDF not designed for fluorescent applications will yield higher background.

Smeary speckles all over the membrane

The speckled, smeary background that frequently obscures western blot membranes is most often due to keratin contamination, a problem that can arise from both the samples being analyzed, and the reagents used throughout the procedure. Keratins are a family of high‑molecular‑weight, highly fibrous structural proteins that are abundant in skin, hair, nails and the outer layers of the laboratory environment itself. Because they are inherently “sticky,” even trace amounts that enter a blot can produce a diffuse, grainy haze or a series of faint, smeared bands that span a wide molecular‑weight range.

Carefully avoid keratin contaminations in your sample and all solutions, buffers and reagents used in your western blot experiment.