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 just look bad- it 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 Western Blot Background

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.

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.
Read more about finding the best blocking agent for your application here.

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 your blot is going to contribute to high background. You may also consider adding additional detergent or changing the detergent in the wash buffer.

4. Find the best exposure time.

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 with a CCD camera makes capturing multiple exposure times even easier.

  • If using an ECL detection system, find a detection reagent with a stable, long-lasting signal (like Radiance ECL) so exposure times are predictable and reproducible, and the signal doesn’t decay so rapidly that you cannot conduct multiple exposures.

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.

In addition to the steps mentioned above, for fluorescent blots there are a couple of additional things to try:

1. You may need to change your membrane.

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

2. Wet membranes also can autofluorescence.

Dry the membrane completely before imaging fluorescent blots.

3. Control the temperature during the 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 Westerns! 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 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.

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 recent work, Mahata et al 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. 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.

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

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.

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 Imager and how Azure can support your research by clicking here.

How to Optimize Your Chemiluminescent Western Blotts

Western Blotting

What is chemiluminescent detection?

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.

How do you use chemiluminescence?

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).

Figure 1.4. 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.

Troubleshooting chemiluminescent Western blots

Because chemiluminescent detection depends on an enzymatic reaction, 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.

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. A longer-lived signal also 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.

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. Tween-20 can cause high background so should be avoided. Anything that impairs enzyme activity or alters the substrate will prevent the production of the light signal. Therefore, all buffers and reagents should be free from substances like azide that inactivate HRP, and the substrate must be protected from heat and light.

Which is better for chemiluminescent blots: film or digital imaging?

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. Importantly, film has a relatively small linear range, so the chemiluminescent signal may become saturated and it might not be possible to capture bright and dim bands with the same exposure.

How to improve your Western blots with digital imaging

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. Finally, digital imaging outputs data that can be directly analyzed using densitometry to obtain quantitative information.

Additional resources

Check out the application note How to Improve Your Chemiluminescent Western Blots to learn more about chemiluminescent blotting. Learn more about the advantages of digital imaging of chemiluminescent Westerns in the application note Why You Should Leave the Darkroom.


Azure offers imaging systems and products to help you achieve the best chemiluminescent Westerns:

• Both the Azure Imagers and the Sapphire Biomolecular Imager include chemiluminescence imaging in addition to many other imaging modalities; find the system that best fits the needs of your laboratory

Radiance chemiluminescent substrate is designed to produce a strong, long-lasting signal for large linear dynamic range and quantitative data

• Azure Chemi Blot Blocking Buffer helps reduce background to improve signal-to-noise ratios


  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.


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!

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.