Common Western Blotting Questions, Answered

Fluorescence imaging Quantification Troubleshooting Western Blotting

Western blotting is a widely used analytical technique that can identify one or more specific proteins in a complex mixture of proteins. It is a powerful tool that provides information about the presence, size, and under the right conditions, even the amount of a protein. Though commonly used and often routine in many labs, Western blotting can be source of frustration when it doesn’t work. It involves several steps (Figure 1), each of which needs to be optimized to achieve the best results. The key to the best Westerns is understanding the process. As a leading manufacturer of Western blot imaging systems, we’re here to help. Here are some answers to your most commonly asked Western blotting questions.

Answers to common Western blotting questions

Several options are available to detect Western blots, with chemiluminescence as a common option. Other means of detection include fluorescence, near-infrared fluorescence, colorimetric, and radioactive.

ExploreWestern Blot Imaging Systems

What reagents do you need for Western bloting?

The reagents needed for Western blotting include a range of essential components, such as enhancing buffers, transfer solutions, stripping buffers, and substrates for fluorescent and chemiluminescent detection. Azure is a one-stop shop for Western blotting, offering reagents and imaging systems for detection of proteins on Western blots. Save the graphic below so you can always make sure you have the correct reagents for your next Western blot.

Western blotting steps with reagents listed
Figure 1. List of comprehensive Western blotting steps with reagents listed

Why are black dots showing on my Western blot?

If black dots are appearing on your Western blot, there may be impurities in the detection antibody you used. You can fix this by filtering the blocking reagent before using. Black dots could also mean there are aggregates in your secondary antibodies; again, filter your secondaries before use. Black dots could also appear due to aggressive stripping techniques.

What is chemiluminescent detection?

Chemiluminescent detection is a method of detecting the location of antibodies bound to a Western blot. Chemiluminescent detection relies on an enzyme, either horseradish peroxidase or alkaline phosphatase, bound to an antibody. The enzyme converts a substrate to a product that emits light (chemiluminescence). The light emitted can be detected using a CCD camera or on X-ray film after processing in a darkroom.

Depiction of chemiluminescent Western blot signal
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.

Developing film can be time consuming, requires access to a dedicated darkroom with appropriate equipment, and necessitates repeated purchase of reagents and single-use film. Digital imaging circumvents the development process altogether and allows labs to leave the darkroom behind. In addition to reducing the waste associated with developing film, digital imaging is more sensitive and provides a larger linear dynamic range than X-ray film. These attributes allow quantitative information to be obtained from Western blots.

What's more sensitive: chemiluminescence or fluorescence?

In general, fluorescent detection can detect picograms of protein while chemiluminescence can detect protein in the femtogram range.

However, sensitivity of detection depends on many things. The ability to detect small amounts of target protein requires a high-quality primary antibody with high affinity and specificity for the target protein. In addition, with CCD cameras, very long exposures are possible to maximize the chance of detecting a low-abundance band but this requires minimizing background “noise” on the Western blot. In addition, different fluorophores have different quantum yields, and some HRP substrates are engineered to increase sensitivity, so the sensitivity of fluorescent detection depends on the specific fluorophore used, and the sensitivity of chemiluminescent detection depends on the substrate used.

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

Continue readingBeginning Chemiluminescent Western Blotting

Shop chemiluminescent substratesRadiance Q

What are the advantages of using fluorescent Western blot vs. chemiluminescent Western blot?

There are many advantages to using fluorescence to detect Western blots over chemiluminescence. The first being that fluorescent Western blotting gives you the ability to multiplex (Figure 2), which uses different fluorescent dyes with non-overlapping excitation and emission spectra, so multiple proteins can be assayed on one blot without needing to strip and re-probe the blot.

Diagram of how fluorescent Western blotting can detect two proteins in two spectrally different channels.
Figure 2. Multiplex detection is possible by using two or more fluorescent dyes and an instrument that can excite and detect the light from each dye.

Fluorescent detection is also more quantitative than its chemiluminescent counterpart when it comes to Western blots. Because chemiluminescent detection relies on an enzyme (HRP or AP) bound to the antibody, the activity of the enzyme can change depending on conditions and as the amount of substrate changes. Fluorescent detection relies on the emission of light from a fluorescent probe bound to the antibody. The fluorescence intensity will only depend on the number of fluorescent molecules present in a given spot.

What is a chemiluminescent substrate?

Chemiluminescent substrates are made of a stable peroxide solution and an enhanced substrate solution that produce light in the presence of HRP and hydrogen peroxide. An example of a chemiluminescent substrate is luminol.

An example of a chemiluminescent substrate is luminol (Figure 3), which is oxidized to 3-aminophthalate which emits light (chemiluminescence) that can be detected using a digital imager with a CCD or CMOS camera, or on X-ray film using a darkroom.

luminol chemical formula
Figure 3. Luminol chemical formula. It is oxidized to 3-aminophthalate which emits light (chemiluminescence) that can be detected on X-ray film or by a CCD camera.

Is HRP a chemiluminescent substrate?

No! HRP is not a chemiluminescent substrate. Even though HRP is an important component of chemiluminescent detection, it stands for horseradish peroxidase. HRP is an enzyme that’s isolated from the roots of the horseradish plant. HRP catalyzes the oxidation of substrates, transferring electrons from the substrate to peroxide. In chemiluminescent Western blot detection, HRP is conjugated to an antibody. The location of the antibody on a blot is then detected by incubating the blot with a substrate that will produce light after it is oxidized by the HRP enzyme.

Diagram illustrating the principles of chemiluminescent Western blotting
The principle of chemiluminescent Western blotting

Azure developed the chemiSOLO to make digital chemiluminescent imaging accessible to every lab. It is a personal Western blot imager that’s able to easily and quickly image chemiluminescent Western blots. chemiSOLO does so without needing a designated laptop of computer- you’re able to use any smartphone or tablet.

chemiSOLO is the first imager of its kind on the market! Get a quote for chemiSOLO by clicking here or filling out the form below. We have an imager for most applications. Explore all imaging systems from Azure Biosystems.

Azure chemisolo next to a hand using a mobile device to connect
A unique web browser interface allows the chemiSOLO to be controlled by phone, tablet, or PC, without the need to install any additional software.

Additional Western Blotting Resources


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!

Problems with Ponceau Stain? Consider Alternative Total Protein Stains for Fluorescent Western Blots

Fluorescence imaging Troubleshooting Western Blotting

Keep reading if you’re having problems using ponceau! Ponceau S is a fast, reversible protein stain often used to confirm that protein samples have successfully transferred from the gel to the membrane before a researcher moves ahead with the time-consuming process of immunoblotting.

Staining the membrane after transfer makes it possible to quickly and easily identify any problems such as incomplete or uneven transfers, or artifacts due to the presence of air bubbles, before probing the blot with antibodies. You can use protein stains to visualize all of the protein bands on a gel or membrane. Staining the blot with some total protein stain can provide a standard for total protein normalization of quantitative Western blots.1

Even though Ponceau staining is reversible, it is not compatible with fluorescent Western blot detection. After thorough destaining, Ponceau stain can leave an autofluorescent residue on the membrane that increases background fluorescence.

  • Azure Quick Tip: To avoid high background due to Ponceau staining, consider using other total protein stains.

    AzureRed Fluorescent Total Protein Stain is completely compatible with downstream Western blotting detection, including fluorescent detection, and with downstream mass spectrometry.

Learn more about AzureRed Fluorescent Total Protein Stain

The figure below shows a multicolor fluorescent Western blot. The right half of the blot was stained using Ponceau and then destained before immunoblotting.

High background when fluorescent western blot stained with ponceau

Both halves of the blot were then blocked with Azure Fluorescent Blot Blocking Buffer and probed for four proteins using four different fluorescent probes as labeled in the figure. The blot was imaged using the Azure Imager RGB module which assesses Cy2, Cy3, and Cy5-compatible channels. Despite thorough destaining, a very high fluorescent High background when NIR western blot stained with ponceaubackground is seen on the half of the blot that was stained with Ponceau.

The figure to the right shows the same blot imaged using the Azure cSeries NIR module, which assesses 700nm and 800nm channels. The fluorescent background is reduced in the NIR-imaged blot, but is still substantial compared to the unstained half of the blot.

If you’re set on using Ponceau, choose Azure Ponceau to detect total protein transferred to membranes. This stain is reversible and can be used to check the efficiency of protein transfer before immunodetection. Each 500 ml contains enough stain for 50 standard blots that are around 9×7 cm.

Shop Ponceau Stain Alternatives, like Azure Ponceau Stain


  1. Thacker JS, et al. Total protein or high-abundance protein: Which offers the best loading control for Western blotting? Biochem. 2016; 496:76-78. PMID: 26706797.
  2. Moritz CP. Tubulin or not tubulin: heading toward total protein staining as loading control in Western blots. 2017;17:1600189. PMID: 28941183.

Sapphire used to Study Anti-cancer Effects in Green Tea

Fluorescence imaging Imaging

Recent work published by Zhao et al in nature communications lends insight into a potential mechanism for the anti-cancer effects of green tea. The health benefits of drinking tea, particularly green tea, has been the subject of intense investigation. A large number of epidemiologic studies have examined whether tea consumption can reduce cancer risk, with a focus on a category of polyphenols called catechins, the most active and abundant of which in green tea is epigallocatechin-3-gallate (EGCG).

Zhao et al demonstrate how EGCG binds to the N-terminal domain of p53 in a recent study. They show that EGCG disrupts the interaction of p53 with MDM2 and inhibits ubiquitylation of p53 by MDM2.

In the publication, ubiquitylation assays were carried out using fluorescein-labeled p53 and varying amounts of EGCG. After incubation with MDM2 and ubiquitin, the amount of free and ubiquitinated p53 was detected on SDS-PAGE gels using the fluorescent channel from an Azure Sapphire Biomolecular Imager (Figure 6A). The amount of ubiquitin was quantified and concentration-dependent inhibition of ubiquitylation by EGCG was analyzed (Figure 6).

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.

Potential anti-tumor properties from green tea

The authors propose the EGCG-induced reduction in ubiquitylation could stabilize p53 protein, making more p53 available and contributing to its anti-tumor activity. Additionally, the work suggests the N-terminal domain of p53 presents an attractive target for anti-cancer drug design.

Fig. 6: EGCG inhibits the MDM2-catalyzed ubiquitination of p53 in vitro.
Figure 6 from Zhao, J., Blayney, A., Liu, X. et al. Nat Commun 12, 986 (2021), licensed under CC BY 4.0. a. In-vitro ubiquitination of fluorescein-tagged p53 was carried out in the presence of up to 0.25 mM EGCG at 20 °C. The results were visualized by fluorescence SDS–PAGE. b. Plotting the fluorescence fraction of residual unmodified p53 as a function of EGCG concentration and fitting to a four-parameter sigmoid indicates that EGCG has an IC50 of ~100 μΜ for the MDM2-catalyzed ubiquitination of p53.

Health benefits of drinking green tea

According to the National Cancer Institute, EGCG and other polyphenols found in green tea have multiple biological activities including being potent antioxidants, inhibiting tumor cell proliferation, inducing tumor cell apoptosis, inhibiting angiogenesis, and activating detoxifying enzymes.

In in-vitro studies, EGCG has been shown to induce apoptosis in cancer cell lines. The transcription factor p53 plays a role in this activity of EGCG. P53 is an important tumor suppressor, promoting cell cycle arrest or apoptosis when a cell is stressed. Usually, cellular p53 levels are low, with p53 being ubiquitylated which targets the protein for degradation. The ubiquitylation is carried out by the protein MDM2, an E3 ligase. When cells are stressed, ubiquitylation is suppressed, p53 is not degraded, and p53 levels increase. The p53 protein then promotes apoptosis through interactions with other proteins and by inducing gene expression changes. Earlier studies have shown EGCG stabilizes p53 and may interfere with the interaction of p53 and MDM2.

Have you published with an Azure instrument?

We’d love to read it! Email your publication to us and we’ll send you something for sharing.

Next time you’re sipping on a cup of green tea with good company, share with them the findings of this study. Science is better shared with a friend! The Sapphire imager provides many imaging capabilities, in addition to multi-channel fluorescence, it can do white light, phosphor imaging, and chemiluminescence imaging of blots, tissues, microplates, and more.

Ready to learn more about the Sapphire imager and how it can support your research? Check out the capabilities of the Sapphire by clicking here.

Sapphire Imager Used in Landmark Study of DNA Repair Pathway

Imaging Protein Assays Transfers

The non-homologous end-joining (NHEJ) pathway is one of two pathways responsible for repairing double-stranded breaks in cellular DNA. Double-stranded breaks can be formed by ionizing radiation or mutagenic chemicals, or as intermediates during normal processes such as the recombination of DNA segments required to create antibody and T cell receptor diversity. In contrast to homologous recombination, in which a double-stranded break is repaired by lining it up with a homologous piece of DNA, NHEJ can ligate any two free pieces of DNA together without requiring extensive sequence homology. Several proteins are known to be involved in NHEJ, but the details of how the complex of these proteins with DNA brings DNA ends together for ligation has been unknown.


Image of PDB ID 7LSY, Chen S, Lee L, Naila T, et al. Structural basis of long-range to short-range synaptic transition in NHEJ. Nature. 2021;78: 2179-2183, created using Mol* D. Sehnal, A.S. Rose, J. Kovca, S.K. Burley, S. Velankar (2018) Mol*: Towards a common library and tools for web molecular graphics MolVA/EuroVis Proceedings. doi:10.2312/molva.20181103), at the RCSB PDB (, H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne. (2000) The Protein Data Bank Nucleic Acids Research, 28: 235-242.

In a new publication in Nature, Chen et al characterize the structure of the protein-DNA complexes involved in NHEJ, and propose a mechanism by which the ends of two DNA molecules are captured by a large complex of proteins and then brought together in a short-range synaptic complex in which the ends are aligned for ligation to close the break. The authors used single particle cryo-electron microscopy to visualize the protein-DNA complexes.

The authors formed the long-range complex by first incubating DNA with the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) followed by the addition of other complex components LigIV-XRCC4 and XLF. Their analysis demonstrated that the resulting 1.66 megadalton structure consists of two complexes of DNA-PK and LigIV-RXCC4, each bound to a DNA end, connected in the middle by one homodimer of XLF. In this complex, the two DNA ends are about 115 angstroms apart, not close enough for ligation.

Comparing the structure to other protein kinases, the authors predicted that the two DNA-PK molecules in the long-range complex are in an active state, and hypothesized that autophosphorylation of each DNA-PK by the other could act as a switch, releasing DNA-PK from the complex. Therefore, they formed a short-range synaptic complex including all of the proteins used earlier except DNA-PKcs. Their structural analyses showed that the resulting complex had a similar overall architecture to the long-range complex but with components shifted and rotated in such a way as to align the DNA ends perfectly for ligation.

The predictions were validated by carrying out in vitro ligation assays in which the Cy5-labeled ligation products were visualized using a Sapphire Biomolecular Imager. No ligation occurred in the long-range synaptic complex without the addition of ATP, an expected result since ATP would be required for the predicted transition to the short-range complex. In contrast, the short-range complex was found to readily ligate DNA, with both strands being ligated more often than single strands.

This important study provides insight into an essential cellular process, and analysis of interactions between components of the NHEJ complexes revealed by the structural study indicate potential mechanisms for some known pathologic mutations and could provide new targets for cancer therapy.

The Sapphire imager provides multiple imaging capabilities including multi-channel fluorescence, white light, phosphor imaging, and chemiluminescence imaging of blots, tissues, microplates, and more. Learn more about the Sapphire imager and how it can support your research by clicking here.

Detecting a fluorescent anti-cancer drug using the Azure c600 imager

Fluorescence imaging

Editor’s note: since the original publishing date of this article, the Azure c600 Imager has been updated to the Azure 600 Imager. Learn more about expanded imaging capabilities of the Azure 600 by clicking here.

In a recent publication, Dostablova et al used the fluorescence imaging capacity of the Azure c600 imager in a creative way, detecting the inherent fluorescence of an anti-cancer drug, as part of their investigation into a novel anti-cancer treatment.

Prostate-Specific Membrane Antigen-Targeted Site-Directed Antibody-Conjugated Apoferritin Nanovehicle Favorably Influences In Vivo Side Effects of Doxorubicin. Dostalova S, et al. Scientific Reports. 11 June 2018. 8:8867. PMID: 29891921.

Doxorubicin-DNA complex Doxorubicin-DNA complex. By Fvasconcellos 19:11, 28 November 2007 (UTC) – From PDB entry 1D12.More information:Frederick CA, Williams LD, Ughetto G, et al. (1990). “Structural comparison of anticancer drug-DNA complexes: adriamycin and daunomycin”. Biochemistry 29 (10): 2538–49. PMID 2334681., Public Domain,

Combating toxicity of anti-cancer therapies

Anti-cancer treatments often are associated with adverse side effects. An approach to reducing the toxicity of anti-cancer drugs is to coat the drug in another substance, called a “nanocarrier.” The nanocarrier can alter drug delivery or absorption, improving delivery to the tumor and/or reducing delivery to other tissues. According to the authors, an ideal nanocarrier is nontoxic, biocompatible, and biodegradable.
Doxorubicin is an anti-cancer drug indicated for the treatment of breast cancer, prostate cancer, leukemia, and many other cancer types. Doxorubicin is associated with multiple adverse effects, the most dangerous of which is dilated cardiomyopathy which can lead to congestive heart failure.

Dostloava et al reported the effects on safety and efficacy of encapsulating doxorubicin with the protein apoferritin (APO). The authors studied both doxorubicin-apoferrin particles (APODOX), as well as a form of APODOX targeted to prostate tumors by conjugating the particles to an anti­–prostate specific membrane antigen (PSMA) antibody (APODOX-anti-PSMA).

Have you published with an Azure instrument?

We’d love to read it! Email your publication to us and we’ll send you something for sharing.

Detecting doxorubicin fluorescence with the Azure c600

In the course of establishing their experimental system, the authors characterized two prostate cancer cell lines to determine whether cellular proteins bound to APODOX-anti-PSMA. They developed an in vitro assay taking advantage of the fluorescence properties of the anti-cancer drug doxorubicin within the APODOX complex. In this assay, similar to a Western blot, cell proteins were separated on a gel and transferred to a PVDF membrane.

The membrane was then incubated with APODOX-anti-PSMA overnight, and washed. Bound APODOX-anti-PSMA was detected by measuring doxorubicin fluorescence using the Azure c600, with an excitation wavelength of 550 nm and emission wavelength of 570 nm. Levels were quantified via densitometry using AzureSpot software.

The APODOX-anti-PSMA was subsequently tested in a mouse model of prostate cancer and the targeted drug was found to attenuate tumors while reducing the damage to kidney and liver tissue that was observed with non-targeted APODOX.