Western Blotting Reagents Roundup – July 2022

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Western Blotting

The Reagent Roundup is made of brief summaries of publications in which researchers used Azure Biosystems reagents for Western blotting and Western blot quantitation in their studies. It is published every quarter. This quarter’s Reagent Roundup features publications from Duke University School of Medicine, University of Minho School of Medicine, the National Institutes of Health, and Boys Town National Research Hospital.

Phosphorylated MED1 links transcription recycling and cancer growth

Aberrant transcription goes hand-in-hand with oncogenesis. Chen et al1 used Chemi Blot Blocking Buffer and Radiance ECL in Western blot experiments investigating transcription recycling in cancer cells. Uncontrolled transcription initiation and elongation are known to be associated with tumor growth but the authors examined whether Pol II recycling, in which RNA polymerase II re-transcribes the same gene rather than being released after transcription is complete, is also associated with cancer. Using a recycling assay developed in their prior publications, the authors demonstrated that Mediator 1 (MED1), when phosphorylated by CDK9, drives Pol II recycling.

Phosphorylation of MED1 increased during prostate cancer progression and inhibiting CDK9 decreased MED1 phosphorylation, Pol II recycling, and prostate tumor growth. The results suggest MED1 phosphorylation and transcription recycling are involved in cancer growth, and MED1 phosphorylation may provide a biomarker to assess therapeutic response of cancers to CDK9 inhibitors.

SHOP: Chemi Blot Blocking BufferRadiance ECL

Aripiprazole Offsets Mutant ATXN3-Induced Motor Dysfunction by Targeting Dopamine D2 and Serotonin 1A and 2A Receptors in C. elegans

Machado-Joseph disease (MJD) is a dominantly inherited progressive ataxia caused by expansion of a CAG repeat in the ataxin-3 gene. Jalles et al2 used Radiance ECL and AzureRed total protein stain, in addition to the Sapphire Biomolecular Imager, in a study investigating how the antipsychotic drug aripiprazole suppresses MJD pathogenesis. In a C elegans model of MJD, the authors found that aripiprazole improved motor performance and this improvement depended on dopamine D2-like and serotonin 5-HT1A and 5-HT2A receptors. Identifying the specific targets of aripiprazole may help develop new therapeutics for MJD with fewer side effects.

DISCOVER: Sapphire Biomolecular Imager

SHOP: Radiance ECLAzureRed

MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization

During metastasis, cancer cells migrate by building out the cytoskeleton at the leading edge of the cell and retracting it at the rear. Pasapera et al3 used Azure’s Radiance ECL in a study of cancer cell cytoskeleton polarization. The authors investigated whether the kinase MARK2, known to regulate the microtubule cytoskeleton in other processes, plays a role in the polarization of the cytoskeleton and directed migration of cancer cells. In osteosarcoma cells, Western blot experiments demonstrated that MARK2 promotes stress fiber formation and myosin II activation and mediates inactivation of myosin phosphatase.

The data suggests MARK2 is a major regulator of cell contractility and adhesion that mediates cancer cell motility.

SHOP: Radiance ECL

Glomerular basement membrane deposition of collagen α1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin α2β1

Alport syndrome is a congenital, progressive glomerular disease that leads to the progressive loss of kidney function. Madison et alused Radiance ECL and TotalStain Q as well as an Azure 600 Imaging System in a study characterizing the glomerular basement membrane (GBM) in a mouse model of Alport syndrome. The investigators found that collagen a1(III) was deposited in the GBM of Alport mice; in wild type mice, collagen a1(III) is found only in the mesangium.

Quantitative Western blotting was carried out using total protein normalization with TotalStain Q staining as the control and the quantitative Westerns confirmed increased levels of collagen a1(III) in the glomeruli of Alport mice. The presence of collagen a1(III) was found to activate DDR1 receptors and lead to changes in gene expression consistent with podocyte injury. Lack of either of the two collagen receptors on podocytes has previously been shown to slow disease progression. The results indicate aberrant collagen-mediated co-receptor signaling through the DDR1 and a2b1 integrin receptors contribute to podocyte injury and renal pathology in Alport syndrome.

DISCOVER: Azure 600 Imaging System

SHOP: Radiance ECLTotalStain Q

Find more publications using Azure reagents and imaging systems on our publications list, or contact us directly for assistance with a specific product by using the form on the left.

Shop Reagents Mentioned

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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!

SOURCES

  1. Chen Z, Ye Z, Soccio RE, et al. Phosphorylated MED1 links transcription recycling and cancer growth. Nuc Acids Res. 2022;500(8):4450-4463.
  2. Jalles A, Vieira C, Pereira-Sousa J, et al. Aripiprazole offsets mutant ATXN3-induced motor dysfunction by targeting dopamine D2 and serotonin 1A and 2A receptors in elegans. Biomedicines. 2022;10(2):370.
  3. Pasapera AM, Heissler SM, Eto M, et al. MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization. Curr Biol. 2022;32(12):2704-2718.
  4. Madison J, Wilhelm K, Meehan DT, et al. Glomerular basement membrane deposition of collagen a1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin a2b J Pathol. 2022; doi: 10.1002/path.5969.

Handy Resources to Improve Western Blots

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Troubleshooting Western Blotting

At Azure Biosystems we live and breathe all things Western blotting. From innovative imagers, to top notch reagents and accessories, to customer education, we are here to support you in your quest for the most robust Western blotting protocols, techniques and data no matter where you are in your Western blotting career.

Are you a Western blotting novice trying to learn more about the process to perfect your technique? Or maybe you’re a veteran Western blotter looking to improve your blots or switch detection methods. In either case, we have resources on Western blotting basics and troubleshooting, along with a team of experts who can help you get your blots just right.

Western Blot imaged with Azure Imager

Helpful resources to help you up your Western blotting game:

Document TypeDescription
App NoteWet or Dry? Which Transfer Method is Best for Your Assay?DOWNLOAD
App NoteHow to Improve Your Chemi BlotsDOWNLOAD
App NoteHow to Improve Your Fluorescence BlotsDOWNLOAD
FlyerTips on Transitioning from Chemiluminescence to FluorescenceDOWNLOAD
App NoteIncreasing Assay Efficiency with Four Color WesternsDOWNLOAD

WATCH: Troubleshooting Common Issues with Western Blotting

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!

Potential Treatment for Advanced Kidney Cancer

Categories
Protein Assays Western Blotting

Renal cell carcinoma (RCC), the most common type of kidney cancer, is aggressive and frequently develops resistance to therapy. Advanced RCC has a poor prognosis and new, effective treatment strategies are badly needed. Metastatic RCC may be treated with sunitinib, but the majority of cancers eventually develop resistance to this drug. Sunitinib inhibits signaling through receptor tyrosine kinases, interfering with pro-growth signals received by the tumor.

In a recent publication, Markowitsch et al investigated the effect of shikonin (SHI) on sunitinib-sensitive and sunitinib-resistant RCC cell lines in cell culture. SHI is a naturally occurring compound. It’s an active component of a dried plant root (Lithospermum erythrorhizon) that has been used in traditional Chinese medicine to address a variety of ailments.

Earlier studies have demonstrated the anti-cancer capabilities of SHI and have shown that SHI can enhance the activity of traditional chemotherapeutics or re-sensitize chemotherapy-resistant cells to therapy. How SHI exerts these effects is not clear; however, as SHI has been found to affect many cell signaling pathways and induce cell death via apoptosis and necroptosis.

The recent work by Markowitsch et al thoroughly examined the effect of SHI on many aspects of RCC cell biology. Several assays relied on imaging with the Azure Sapphire Biomolecular Imager, including studies that characterized protein expression related to multiple signaling pathways by Western blot, adhesion of RCC cells to extracellular matrix proteins and to vascular endothelial cells, and studies of tumor cell migration and chemotaxis, relied on imaging with the Sapphire.

Figure 2 from Markowitsch et al. (2022) Shikonin inhibits cell growth of sunitinib-resistant renal cell carcinoma by activating the necrosome complex and inhibiting the AKT/mTOR signaling pathway. Licensed under CC BY 4.0. The Azure Biomolecular Imager was used to image and quantify RCC colonies on cell culture dishes.

The authors took advantage of several imaging modes provided by the Sapphire. The Sapphire was used to image and quantify the growth of colonies in 6-well culture dishes using Coomassie Blue dye detection. The Sapphire was used to assess cell adhesion, chemotaxis and cell motility by measuring the fluorescence of cells labeled with CellTracker Green in either pre-treated 24-well culture dishes or on the lower surface of membrane inserts in 24-well plates. Western blots were detected using enhanced chemiluminescence and imaged on the Sapphire.

The results of the numerous studies indicated that, though the specific effects varied by cell line, SHI had antitumor effects on all cell lines studied. SHI was found to inhibit RCC cell growth, proliferation, and clone formation, both in sunitinib-sensitive and sunitinib-resistant cell lines. SHI caused cell cycle arrest and induced cell death, primarily via necroptosis. SHI also inhibited the AKT/mTOR pathway, which presents another mechanism by which SHI may interfere with RCC cell survival and growth.

The authors conclude the data is promising and that SHI should be further studied as a potential addition to therapy for patients with advanced and therapy-resistant RCC.

In addition to visible and fluorescent imaging of tissue culture plates, and chemiluminescent imaging of Western blots, the Sapphire Biomolecular Imager provides densitometry, phosphor, multichannel fluorescence, 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.

 

Explore: Azure Sapphire Biomolecular Imager

Which Imager Should You Choose for Total Protein Normalization?

Categories
Fluorescence imaging Imaging Troubleshooting Western Blotting
Total protein normalization (TPN) is used to quantify the abundance of the protein of interest, without having to rely on housekeeping genes. It is usually done by incubating the membrane with a total protein stain. 
TPN has become the preferred method for normalizing Western blot data. But to fully leverage its benefits, you need an imaging system that allows you to multiplex TPN with detection of your target proteins. Fortunately, we have just what you need! Whether you want to combine TPN with near infrared (NIR) fluorescent Western blotting, or with enhanced chemiluminescent (ECL) detection, read on to learn how Azure imaging systems enable you to do so with ease.

Why you should use Total Protein Normalization (TPN)

A major advantage of TPN is that it delivers more accurate quantification of target analytes than the established practice of using individual housekeeping proteins. This is because TPN is less susceptible to change in response to experimental treatments, providing a more reliable baseline against which target protein expression can be compared. TPN also avoids the problem of over-saturation where low abundance analytes require high protein loads to reach the necessary sensitivity for detection since it has an incredibly broad dynamic range (1–50 μg of lysate).

How is TPN currently performed?

Current methods for TPN vary according to the chosen readout. Where TPN is combined with ECL detection, it is common practice to use specialized gels that chemically modify all of the proteins within each sample upon exposure of the gel to UV light, enabling their subsequent measurement. A drawback of this approach is that it has only a narrow range in which the protein load is linear.

In situations where TPN and NIR detection are paired, two distinct techniques are used. The first involves labeling the entire protein population of each sample with a fluorescent dye before loading the gel, a process that introduces an additional source of variability to the workflow. The second requires that the membrane be stained with a NIR reagent for TPN immediately after transfer; the membrane is then imaged and de-stained prior to NIR target detection. Because de-staining is never 100% complete, this latter method essentially restricts target detection to just one of the two available NIR channels.

How does Azure's approach to TPN improve on existing methods?

Azure’s range of imaging systems includes several models that allow target protein detection to be multiplexed with TPN – with no need for dedicated precast gels or laborious stripping and re-probing. Instead, you simply treat your blots with our total protein stain – TotalStain Q – between protein transfer and blocking, and process them as you would normally.

Which Azure imagers support TPN?

For TPN with NIR Western blot detection, the Azure 500Q and the Sapphire NIR-Q both provide detection of target analytes in the 700nm and 800nm channels, with a third (520nm) channel used to measure TotalStain Q. By reserving the NIR channels for your proteins of interest, sensitivity is uncompromised by integrating TPN into your Western blotting workflow.

Azure imaging systems enable target protein detection to be multiplexed with TPN. The Azure 300Q, Azure 500Q and the Sapphire NIR-Q all include the Q module (our optional green fluorescence channel) to quantify total protein staining.

Where TPN and ECL are performed in parallel, the Azure 300Q is a compact benchtop solution that can readily be upgraded to include visible and/or NIR fluorescent detection capabilities as your Western blotting requirements evolve. And if you already have an Azure 300 or Azure 500 in your lab, adding our optional green fluorescence channel – the Q module – to your system means you can easily begin multiplexing TPN without interruption to your Western blotting workflow.

Want to find out how you can add multiplex total protein normalization with NIR fluorescent Western blot detection or ECL to your research? Send us a message using the form on this page. 

BONUS: We’re giving away free samples of TotalStain Q – our newest reagent for total protein staining! Grab a sample!

If you’d like to learn more about how TPN can enhance your Western blotting data, check out this webinar:

Help! Why do my Western blots look terrible?

Categories
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 Background in Western Blots

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.

Finding the best blocking agent for your application by clicking 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 the 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 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 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.

Additional Things to Keep in Mind for Fluorescent Blots

Change your membrane

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

Remember that wet membranes also can autofluorescence

Dry the membrane completely before imaging.

Be sure to 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 Westerns! If you still have questions, fill out the form on the right and one of our experts will reach out to assist.

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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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Regulation of Gene Expression by Enhancer RNAs

Categories
Imaging Quantification Transfers Western Blotting

The regulation of gene expression is a complicated affair. A vast array of control mechanisms exist that can adjust the levels of gene expression products to match the needs of the cell. Messenger RNA (mRNA) can be processed to alter its stability, protein translation from mRNA can be controlled, and the stability and/or activity of the protein can be altered via post-translational modifications. The first point of control of gene expression is the initiation of gene transcription.

Transcription initiation has several requirements. The chromatin structure must open to make the gene accessible to the transcriptional machinery. In eukaryotic cells, the promoter sequence of the gene must be bound by transcription factors that direct RNA polymerase to the gene to begin transcription. Transcription initiation is made more likely by the binding of activator proteins to other DNA regions near the promoter called enhancers, which can be located up- or downstream of the transcription start site.

Genome-wide sequencing experiments have revealed that RNA molecules are transcribed from many enhancer regions, indicating the enhancer regions may not merely be binding sites for activator proteins. These enhancer RNAs (eRNAs) are non-coding RNAs (ncRNAs) and are not translated into proteins. It is possible that eRNAs may simply be the result of non-specific transcription by RNA polymerase and serve as a sign that chromatin is open and accessible to RNA polymerase in a region or DNA. Alternatively, there is evidence some eRNAs may serve an active role in regulating gene expression by themselves binding to and changing the activity of proteins.

In recent work, Setten et al studied an eRNA transcribed from an enhancer near the gene encoding a transcription factor called CEBPA (CCAAT enhancer-binding protein alpha). CEBPA was of interest because this transcription factor is involved in many processes, including cell cycle inhibition and tumorigenesis, and because it is expressed in specific cell lineage and plays a role in maintaining cell identity. The researchers set out to determine whether an eRNA transcribed from an enhancer 9kb downstream from the transcription start site of the human CEBPA gene was involved in regulating CEBPA expression. They called this eRNA CRED9.

Figure 7 from Setten RL, Chomchan P, Epps EW, et al. (2021) CRED9: A differentially expressed elncRNA regulates expression of transcription factor CEBPA. Licensed under CC BY 4.0. Quantitative fluorescent Western blot showing levels of CEBPA isoforms detected with a goat–anti rabbit 800 secondary antibody from Azure Biosystems (red) and a NIR protein ladder (blue) (panel A). Before blocking the membrane was stained with a NIR fluorescent total protein stain and an image acquired for total protein normalization (panel B).

The authors found that levels of CEBPA mRNA and the CRED9 eRNA were correlated across several different cell lines; when CRED9 was high, CEBPA mRNA was also high. They then knocked down CRED9 in a cell line and found that when CRED9 levels were reduced, CEBPA mRNA and CEBPA protein levels were also reduced. Finally, knockdown of CRED9 reduced the amount of a histone H3K27ac bound to the enhancer, indicating that the activity of the enhancer region was reduced. These results lead the authors to propose that CRED9 and other eRNAs may have an active role in enhancer function and gene regulation.

To study CEBPA protein levels, the authors carried out a quantitative near-infrared fluorescent Western blot imaged on an Azure Sapphire™ Biomolecular Imager. To quantify changes in protein level between samples, the fluorescent signal from the Western blot was normalized to the total protein loaded, as was visualized on the Sapphire Biomolecular Imager.

In addition to multichannel and NIR fluorescent imaging, the Sapphire Biomolecular Imager provides chemiluminescence, densitometry, phosphor 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.

Investigating S-acylation of the SARS-Cov-2 Spike Protein Leads to New Insights into Viral Infectivity

Categories
COVID-19 Fluorescence imaging Imaging Western Blotting

A better understanding of coronavirus biology can enable development of new antivirals to help stop COVID-19 and prevent future pandemics. In recent work, Puthenveetil et al. characterized S-acylation of the Spike (S) protein of SARS-CoV-2. This post-translational modification is known to be important to the viral replication cycle of other viruses across multiple virus families but has not been studied in SARS-CoV-2.

S-acylation involves adding long-chain fatty acids to cysteine residues on the cytosolic side of transmembrane proteins. The cytoplasmic tail of the SARS-CoV-2 S protein contains 10 cysteines in 6 potential S-acylation sites. Puthenveetil et al note that all but one of these are conserved with SARS-CoV, and most are conserved with other coronaviruses that infect humans, including MERS. Still, nothing is known about what role, if any, the S-acylation of these cysteines may play in the biology of these viruses.

To answer that question, the authors expressed S protein in cultured cells and carried out metabolic labeling with a fatty acid that was detected using a rhodamine-labeled fluorescent probe. They found substantial S-acylation that was blocked by 2BP, a global inhibitor of S-acylation. The authors also found the S-acylation of the S protein was dependent on the presence of the cysteines in the C-terminal domain.

Further experiments expressing mutated versions of the S protein identified which cysteines were S-acylated, the effect of mutating these cysteines on particle infectivity, and which members of the human family of enzymes that carry out S-acylation were able to modify the S protein in cells and in an in vitro assay.

To control for gel loading and S protein expression in the S-acylation experiments, the authors used the Azure Sapphire Biomolecular Imager to detect GAPDH by NIR fluorescence and SARS-CoV-2 S protein by chemiluminescence on Western blots.

In addition to chemiluminescence and near-infrared fluorescence imaging, the Sapphire provides densitometry, phosphor, multichannel fluorescence, 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 hereHave you published with one of our instruments? We’d love to read it! Send a link to your publication to info@azurebiosystems.com– we’ll send you something for sharing.

Multiplex Westerns – Insider tips

Categories
Multiplex Western Blotting

Detecting one protein, two proteins, three proteins? How about FOUR proteins on the same Western Blot? Save money on each experiment by using multiplex Western blots.

Here at Azure Biosystems we’re great believers in the power of fluorescent multiplexing in Western blots, which has driven a lot of the development of our line of Azure imagers. But we recognize that taking the step from a standard HRP/chemiluminescent-based approach can be daunting. Keep reading for 6 tips to make the switch as easy as possible.

Are you having problems with your multiplex fluorescent Western blots? In this post, we’ll cover the exact protocols in more detail in some of our application notes covering phosphorylation/total protein detection, and single step normalization.

1. Increase your concentrations

The concentration of both the primary and secondary antibody required may be increased compared to chemiluminescent Westerns depending which imager you’re using. In more modern or laser based imagers, this effect may be less marked.

2. Test individually first

Going hand in hand with the above point, it always makes sense to test your antibodies individually first. That way you can determine optimal concentrations, factors contributing to background or lack of specificity in a simpler environment, rather than trying to unpick 3 different primary and secondary antibodies at once.

3. Use adsorbed secondary antibodies

Although it sounds simple, many people don’t consider that they are now adding multiple antibodies from multiple species onto a single blot. If you work with multiple fluorescence in other fields you’ll already be aware of the importance of cross-adsorbing secondary antibodies to reduce inter-species cross reactivity. But many people don’t consider this for Westerns, it’s worth checking out your antibodies to ensure they meet the grade.

4. Expand the spectrum

Three-color Westerns are exciting, but what about five colors? With NIR capability the number of spectrally distinct peaks that can be isolated can be increased greatly. Obviously, this may require a bit of work up to get the antibodies optimized, but imagine the time and cost savings generated by performing one Western for five proteins.

5. Check your membrane

Some membranes will auto-fluoresce when exposed to UV light generating a high background signal, although more and more fluorescent safe membranes are being developed. We would also recommend switching to using PVDF membranes from nitrocellulose due to its increased sensitivity, as we discussed previously.

BUY: PVDF membanes

6. Choose the right channel for your protein

Unfortunately all detection channels were not created equally. For standard fluorescence use blue to detect your highest abundance protein, green the middle and red for your lowest abundance protein. If introducing NIR the excellent sensitivity and low background achieved with these fluorophores also makes them ideal for low abundance proteins.

Have more questions or want to learn more about multiplexing and how it can improve the way you research? Fill out the form on the left- we’ll be in touch.

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How to Optimize Your Chemiluminescent Western Blotts

Categories
Western Blotting

What is chemiluminescent detection?

The first step to troubleshooting chemiluminescent Westerns is understanding what they are. You’re in the right place! 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).

chemiluminescent western blot signal
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 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. Now, let’s try to troubleshoot your chemiluminescent Western blot:

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 for troubleshooting chemiluminescent Western blots

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

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.

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!

Studying the SARS-CoV-2 proteases with the Sapphire Biomolecular Imager

Categories
Fluorescence imaging

Researchers at the Wroclaw University of Science and Technology and their colleagues have recently published exciting work improving our understanding of the two proteases encoded by the SARS-CoV-2 genome, the main protease (Mpro) and the papain-like protease (PLpro). These proteases are required for viral gene expression and for viral replication, so they are attractive targets for antiviral drug development. In addition, PLpro can inhibit the host innate immune response via deubiquitination activity, so inhibition of this protease may provide another mechanism of antiviral activity.

In each publication, the scientists used hybrid combinatorial substrate libraries to understand the rules governing substrate specificity of these proteases. Then, these rules guided the design of activity-based probes and irreversible inhibitors for each protease.

To characterize binding of their compounds to the proteases and the deubiquitinase activity of PLpro, the authors took full advantage of the fluorescence detection capabilities of the Sapphire Biomolecular Imager.

In Science Advances, the group examined SARS-CoV-2 PLpro, designing and characterizing two inhibitors. Using a biotinylated ubiquitinated probe, they found no evidence for cross-reactivity between their inhibitors and human deubiquitinases, an important requirement for potential antiviral drugs targeted to this protease. The biotinylated probe, bound to proteins in cell lysates, was detected on blots using an Alexa Fluor 647¬–labeled streptavidin as a probe and imaging the blots with the Sapphire Imager. To study the deubiquitinase activity of SARS-CoV-2 PLpro, binding and cleavage of ubiquitin-containing substrates was detected in SYPRO Ruby-stained gels, also imaged using the Sapphire Imager.

In Nature Chemical Biology, the group focused on SARS-CoV-2 Mpro, designing an inhibitor that demonstrated significant antiviral activity against SARS-CoV-2 in a cell culture assay, and an activity-based fluorescent probe that could be used to visualize active Mpro in cells taken from patients with COVID-19 infection. To study the binding of their compounds to SARS-CoV-2 Mpro, compounds labeled with Cy5 or BODIPY were detected directly on blots. Biotinylated versions were detected using Alexa Fluor 647¬–labeled streptavidin as a probe.

This research spotlights numerous avenues for development of potential anti-coronavirus drugs and tools for disease characterization and diagnostics.

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https://doi.org/10.1126/sciadv.abd4596
https://doi.org/10.1038/s41589-020-00689-z

Gruber, Christian C.; Steinkellner, Georg (2020): Comparative model of novel coronavirus 2019-nCoV protease Mpro. figshare. Dataset. https://doi.org/10.6084/m9.figshare.11752752.v5 licensed under CC BY 4.0.