What is Western Blotting?

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Fluorescence imaging Quantification Troubleshooting Western Blotting

Your common Western blotting questions, answered.

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, each of which needs to be optimized to achieve the best results. The key to the best Westerns is understanding the process. We’re here to help with some answers to your most commonly asked Western blotting questions.

What are some of the detection methods used in Western blotting?

Several options are available to detect Western blots. Chemiluminescence is likely the most common. Other means of detection include fluorescence, near-infrared fluorescence, colorimetric, and radioactive.

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What is chemiluminescent detection?

In Western blotting, 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 on X-ray film or by CCD camera.

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

Continue readingBeginning Chemiluminescent Western Blotting
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What are the advantages of using fluorescent Western blot vs. chemiluminescent Western blot?

Multiplex detection is possible by using two or more fluorescent dyes on an instrument that can excite and detect light from each dye
Multiplex detection is possible by using two or more fluorescent dyes and an instrument that can excite and detect the light from each dye.

1. Fluorescent Western blotting allows multiplexing. By using different fluorescent dyes with non-overlapping excitation and emission spectra, multiple proteins can be assayed on one blot without needing to strip and re-probe the blot.

2. Fluorescent detection is more quantitative than chemiluminescent detection. Chemiluminescent detection relies on an enzyme (HRP or AP) bound to the antibody, and 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.

Continue readingAlternative Total Protein Stains for Fluorescent Western Blots

Is HRP a chemiluminescent substrate?

The principle of chemiluminescent Western blotting
The principle of chemiluminescent Western blotting

No! Even though it is an important component of chemiluminescent detection, HRP stands for horseradish peroxidase, an enzyme 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 and the location of the antibody on a blot is detected by incubating the blot with a substrate that will produce light after it is oxidized by the HRP enzyme.

ExploreHRP Stripping Buffer

What is a chemiluminescence substrate?

luminol chemical formula
Luminol chemical formula

Chemiluminescent substrates produce light in the presence of HRP and hydrogen peroxide. An example of a chemiluminescent substrate is luminol. Luminol is oxidized to 3-aminophthalate which emits light (chemiluminescence) that can be detected on X-ray film or by a CCD camera.

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 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 Western blotting experts will reach out to assist.

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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.

Secrets to Chemiluminescent Western Blotting

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Western Blotting
What you need to know to optimize your chemiluminescent Western blots

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).

Chemiluminescent Western Blot
Chemiluminescent Western Blot Detection With HRP

Troubleshooting chemiluminescent Westerns

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.

Digital imaging using a CCD camera provides 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.

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

SOURCE
  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.

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!