Common Western Blotting Questions, Answered

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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

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Help! Why do my Western blots look terrible?

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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.

FREE WESTERN BLOT eBOOK

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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How to Optimize Your Chemiluminescent Western Blots

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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).

SOURCES

  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

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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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