Help! Why do my Western blots look terrible?

Western Blotting

One of the most common questions when troubleshooting problematic Western blots is, “Why is the background so high?”

High or uneven background doesn’t only look bad- it also interferes with data analysis, making it difficult to quantify bands or compare bands between samples. There are several things you can do to reduce background and increase the signal-to-noise ratio on your blots. Read on for steps to help you achieve high-quality data and publication-worthy images!

5 Steps to Reducing Background in Western Blots

STEP 1: Use clean, fresh buffers

Make sure your blotting and wash buffers are made fresh. You may want to filter them to remove dust or particulates that may be deposited on your blot and interact with your antibodies or other components of the blotting protocol.

STEP 2: Use the correct blocking agent

Make sure you select a blocking agent that doesn’t interact with your antibody or block your epitope! Commonly used protein-based blocking agents can be problematic in specific situations, particularly with anti-phosphoprotein antibodies.

Unsure which blocking buffer to use? Click below for a free sample.

STEP 3: Don’t skimp on the wash steps!

Make sure you use sufficient wash buffer, wash for a long enough time, and agitate the membrane well during wash steps. Any non-specifically bound antibody left on the blot is going to contribute to high background. You may also consider adding additional detergent or changing the detergent in the wash buffer.

STEP 4: Find the best exposure time for your chosen detection method

When over-exposed, any blot can appear as solid background. Ideally, the signal from specific bands is much stronger than any background noise and a short exposure will pick up only the specific signal. If using film, be prepared to expose the blot multiple times to different pieces of film for increasing periods of time to find the optimal exposure.

Imaging using an imager like the Azure 600, or another digital imager with a CCD camera makes capturing multiple exposure times even easier.

Azure 600 Western blot Imaging system
The Azure 600 is the only system that offers two channel, laser-based IR and chemiluminescent detection, with the speed and sensitivity of film, with the ability to image visible fluorescent dyes, standard EtBr and protein gels, and infrared laser excitation for quantitative Western blot imaging in the NIR. This catchall Western blot imager improves your data quality imaging with infrared dyes and offers signal stability.

If you’re using an ECL detection system, use Radiance ECL, a detection reagent with a stable, long-lasting signal, so exposure times are predictable and reproducible. Using Radiance helps ensure the signal doesn’t decay so rapidly that you cannot conduct multiple exposures.

STEP 5: Optimize your antibody concentrations

This is a situation where some initial work up front can save you a lot of time down the line. Using too much antibody can increase the amount of antibody that binds non-specifically to the membrane. Start with the antibody dilution recommended by the antibody provider.

  • If background is high, dilute the antibody more, increasing the incubation time if necessary.
  • Incubating at 4 °C can also help reduce non-specific binding.

Quick Tips to Keep in Mind for Fluorescent Western Blots

  • Azure Quick Tip #1: Change your membrane

    Nitrocellulose and some PVDF membranes can autofluoresce. To reduce background from your membrane, use only low-fluorescence PVDF membranes.

  • Azure Quick Tip #2: Remember that wet membranes can also autofluorescence

    Dry the membrane completely before imaging.

  • Azure Quick Tip #3: Control the temperature during the protein transfer step

    Excessive heat during transfer is usually a major source of background in fluorescent Western blotting.

With these tips, you’re on your way to reducing the background and getting clean, clear Western blots. If you still have questions, fill out the form on the right and one of our experts will reach out to assist.


New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices? Claim your free Western Blotting eBook!
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A New Mechanism by Which Bacteriophage T5 Inhibits Growth of E. coli

Protein Assays Publication Spotlight Quantification

How can we better control pathogenic bacteria? Insights may come from studying bacteriophages, viruses that infect bacteria. There are a wide variety of bacteriophages, each of which is specialized to infect and replicate within a specific target bacteria. Learning how a bacteriophage takes over bacterial metabolism to direct resources towards generating more bacteriophage can both increase understanding of bacterial metabolism, and potentially provide ideas for new antibiotics or new means of controlling bacterial pathogens.

In recent work, Mahata et al from Tel Aviv University in Israel developed a high-throughput sequencing approach to identify functions for T5 proteins. The bacteria were mutagenized and then screened to identify bacterial mutants that were resistant to growth inhibition by the phage protein T5.015.

To demonstrate the DNA cleavage activity of T5.015, the Azure Sapphire™ Biomolecular Imager was used to detect cleavage products of Cy5.5-labeled oligomers separated by gel electrophoresis (Figure 1). High-throughput sequencing of the mutants characterized the DNA changes responsible for the resistance. The researchers found mutations in the ung gene made the bacteria resistant to the effects of T5.015.

Cy5.5 fluorescence of assay products from Azure Sapphire Biomolecular Imager resolved on denaturing urea polyacrylamide gel are shown.
Figure 1. 015 cleaves Ung-generated AP sites. (A) Schematic diagram of the Ung-generated AP site cleavage assay. (B and C) AP site cleavage assay performed in the presence of the Cy5.5-labeled dsDNA oligomer and the indicated purified proteins. In B, 33.4 nM (1 U) of Ung, 6.6 nM (2 U) of Ape1, 1.26 µM of 015, and 820 nM (2 U) of Ugi were used. In C, 315 nM of 015 and 6.6 nM of Ape1 were used. Cy5.5 fluorescence of assay products resolved on denaturing urea polyacrylamide gel are shown.

Ung, the protein encoded by the ung gene, is involved in uracil excision, removing uracils mistakenly incorporated into DNA. Normally the Ung protein removes the uracil, and the resulting abasic site in the DNA is repaired. However, the researchers found that in T5 infection, after Ung removes an uracil, T5.015 cleaves the DNA at the abasic site. DNA cleavage pauses DNA replication and inhibits bacterial growth. The authors hypothesize that halting DNA replication and cell division makes more resources available to the phage.

Conveniently,­ T5 encodes a dUTPase that reduces UTP levels in the bacteria after infection so newly synthesized phage DNA is much less likely to contain any uracil, and only bacterial DNA is targeted by T5.015. The mechanism identified by Mahata et al represents a previously unknown means of bacterial growth inhibition by a bacteriophage.

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Model of 015-mediated toxicity. T5 bacteriophage translocates its genetic material into the E. coli cell, which expresses 015 within several minutes (1). The 015 gene product forms a complex with the host Ung (2) and thus localizes to newly formed AP sites (3). 015 then attacks the AP site (4) and forms a nick in the chromosomal DNA (5), which leads to DNA replication arrest and ultimately to cell death (6). Licensed under CC BY 4.0

Bacteriophage T5 infects the bacteria Escherichia coli. Some strains of E. coli are found normally in the human gut, but other strains are pathogenic and are responsible for some cases of foodborne illness. T5 is an intriguing bacteriophage because of its large genome which encodes over 160 proteins, only about half of which have known, or proposed functions based on homology. Therefore, studying T5 holds the potential to reveal novel bacterial biological or biochemical mechanisms in addition to providing potential new avenues to controlling pathogens.

In addition to multichannel fluorescent imaging, the Sapphire Biomolecular Imager provides chemiluminescence, densitometry, phosphor, near-infrared and white light imaging of blots, gels, tissues, and more. Learn more about the Sapphire by clicking here and requesting a demo.

How to Optimize Your Chemiluminescent Western Blots

Western Blotting

Chemiluminescent detection (Figure 1) depends on an enzymatic reaction so timing and the amount of both enzyme and substrate used have important effects on data quality. Light will only be produced while the enzyme has access to the substrate, so the blot must be imaged before the substrate is consumed and before the light signal decays. The exposure time needed to detect the signal increases as the signal declines over several minutes, leading researchers to conduct multiple exposures to try to capture the perfect image before the signal decays.

Depiction of chemiluminescent Western blot signal
Figure 1. Chemiluminescent Western blotting- one signal, one protein. In chemiluminescent detection, the antigen-primary antibody complex is bound by a secondary antibody conjugated to an enzyme, such as horseradish peroxidase (HRP). The enzyme catalyzes a reaction that generates light in the presence of a luminescent substrate, and the light can be detected either by exposure to x-ray film or by a CCD-based imaging system.

Is film or digital imaging better for chemiluminescent Western blotting?

The chemiluminescent signal is usually detected either by exposing the blot to film, or by using a CCD camera. Film is expensive due to the cost of the film and of the reagents and equipment needed for developing. Film has a relatively small linear range, so the chemiluminescent signal may become saturated. It might not be possible to capture bright and dim bands with the same exposure.

Are you looking for an affordable option to quickly image Western blots? The chemiSOLO is a newly launched, personal Western blot imager that’s capable of detecting low-expressing proteins with femtogram sensitivity. chemiSOLO is able to capture marker images at the push of a button. Learn more about how to easily capture chemiluminescent Western blot images using the new chemiSOLO by clicking here.

Connecting laptop to Azure chemiSOLO chemiluminescent western blotting imager
chemiSOLO is a personal Western blot imager used to capture pictures of colorimetric blots or visible-stained protein gels, like Coomassie blue or silver stain. A unique web browser interface allows the chemiSOLO to be controlled by phone, tablet, or PC, without the need to install any additional software, making it a versatile imager for chemiluminescent Western blotting.

Why is the background on my Western blot so high? Why is there low (or no) signal?

Using too much secondary antibody can result in high background due to excess antibody binding nonspecifically to the blot. Too much secondary antibody (or too little substrate) can also reduce sensitivity because substrate will be used up too quickly and the light signal may decay before imaging can be conducted.

Keep in mind that other buffer components used in washes or to dilute components can affect the reaction. Anything that impairs enzyme activity or alters the substrate will prevent the production of the light signal. Avoid using Tween-20, as it can cause high background. Instead, use Chemi Blot Blocking Buffer to help reduce background and improve signal-to-noise ratios on your Western blot.

Best substrate to use for chemiluminescent Western blots

Some commercial substrates are modified to extend the lifespan of the light signal to hours rather than minutes, which can provide the researcher with more flexibility when imaging. That’s where Radiance comes in. Radiance is a specially formulated, chemiluminescent substrate designed to produce a strong, long-lasting signal for large linear dynamic range and quantitative data.

A longer-lived signal improves reproducibility between experiments because the signal remains constant for a longer period of time, reducing the effect of slight differences in elapsed time between substrate incubation and imaging.

  • Quick Tip: All buffers and reagents should be free from substances like azide that inactivate HRP.

    The substrate must be protected from heat and light.

Digital imagers for the best chemiluminescent Western blots

Digital imagers that use a CCD camera provide a larger dynamic range, overcoming this limitation of film. Digital imaging saves time, giving instant results so researchers can quickly determine whether the selected exposure time is sufficient rather than waiting several minutes to develop film, during which time the chemiluminescent signal may be decaying. Digital imaging outputs data that can be directly analyzed using densitometry to obtain quantitative information.

Azure Imagers also allow you to use multiple binning options to collect more light. Both the Azure Imagers and the new Sapphire FL Biomolecular Imager include options for chemiluminescent Western blot imaging, in addition to many other imaging modalities; find the system that best fits the needs of your lab by clicking below.

What is chemiluminescent detection?

With chemiluminescent detection, a primary antibody binds to the target protein on a membrane, and the location of the primary antibody is detected using a secondary antibody conjugated to an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP).

A substrate for the enzyme is added and when the enzyme acts on the substrate, light is emitted (Figure 1). The light can be detected using an imager with a CCD camera or x-ray film. The sensitivity of detection depends on the choice of substrate—commercially available substrates for HRP can detect proteins in the femtogram range.

LEARN MORE: Check out this application note How to Improve Your Chemiluminescent Western Blots to learn more about chemiluminescent Western blotting. If you want to learn more about the advantages of digital imaging of chemiluminescent Westerns read Why You Should Leave the Darkroom.

chemiluminescent western blot signal
Figure 1. Chemiluminescent Western blotting- one signal, one protein.

How do you use chemiluminescence to detect proteins?

Chemiluminescence remains the most frequently used method to detect target proteins on Western blots. Many reagents are commercially available for chemiluminescent detection but all share basic characteristics. The secondary antibody is labeled with an enzyme, usually horseradish peroxidase (HRP). After incubation with the secondary antibody, the membrane is incubated in a solution containing a chemiluminescent HRP substrate such as luminol.

When HRP reacts with the substrate, light is produced (Figure 1). Most commercial substrates also contain additional compounds that increase and stabilize the light signal, providing enhanced chemiluminescence (ECL).


  1. Alegria-Schaffer A, Lodge A, Vattem K. Chapter 33. Performing and Optimizing Western Blots with an Emphasis on Chemiluminescent Detection. Methods in Enzymology. Vol 463. 2009, Elsevier Inc.

  2.  Mruk DD, Cheng CY. Enhanced chemiluminescence (ECL) for routine immunoblotting; an inexpensive alternative to commercially available kits. Spermatogenesis. 2011;1(2):121-122.


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Studying Tissue Morphology with the Sapphire Biomolecular Imager

Fluorescence imaging
Sheep kidney imaged using 488nm and 658 nm lasers, 10 micron resolution

The Sapphire Biomolecular Imager can do so much more than image gels, blots, and microwell plates. With its 25 cm x 25 cm scanning bed, the versatile Sapphire can scan tissues and even small animal models such as mice, zebrafish, and Xenopus oocytes, to study tissue morphology or gross anatomy.

Bakela et al took advantage of this capability of the Sapphire Imager to study liver morphology in a recent publication. The group investigated the ability of soluble major histocompatibility complex II (sMHCII) molecules to rescue symptoms of autoimmune hepatitis (AIH) in a rat model of the disease.

Chronic AIH is characterized by a T-cell-mediated autoimmune response that attacks the liver. The disease is usually treated with immunosuppressive drugs. New and specific therapies are needed to better treat the disease and to avoid the side effects associated with long-term use of immunosuppressants.

The authors set out to test whether sMCHII molecules could rescue liver damage in a rat model of AIH. These molecules are hypothesized to help maintain immune tolerance and promote immune system suppression, protecting against autoimmunity. Promisingly, sMCHII molecules had been tested previously in a model of systemic lupus erythematosus and found to decrease the amount of autoantibodies and improve symptoms.

To characterize the liver damage that occurred in the AIH rat model, the authors collected and fixed livers from the rats and then scanned them on a Sapphire Biomolecular Imager using white light as well as four-channel fluorescence. The four-channel images, detecting tissue autofluorescence, provided detail of the gross anatomy and morphology of the liver tissue. Treatment with sMCHII appeared to rescue the fibrotic and necrotic changes that were observed in the livers of untreated rats, leading the authors to propose this approach could lead to new therapies for AIH.

Learn more about applications of the Sapphire Biomolecular Imager, including scanning tissues and small animal models using fluorescence, chemiluminescence, and phosphorimaging, here.

Since the release of this publication, the Azure Sapphire has been succeeded by the new Azure Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more.

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