Author: Tamara

Quantitative Westerns: What is the Best Way to Normalize your Western blot?

Categories
Fluorescence imaging Multiplex Quantification Western Blotting

Far from being an “is-it-there-or-not” technique, modern digital detection instruments can make Western blotting reproducible and quantitative. By working within the linear dynamic range of your detection method and normalizing the data to control for variations in protein load and membrane transfer, you can get truly quantitative results.

But what is the best way to normalize protein levels for a Western blot? In the past, the gold standard normalization method was to use a housekeeping protein based on the assumption that the levels of these proteins are fairly consistent across experimental conditions and cell lines. However more recent studies have shown that this assumption is not always true1,2 leading to inaccurate measurements of relative protein abundance. Instead, quantitative Western blotting experts1,2 and the journals they publish in4 are recommending a new gold standard for normalization—normalizing to total protein detected in each lane, preferably by staining on the membrane.

Table of contents

Using Total Protein Stains for Normalization

With total protein normalization, instead of trying to find a protein that can represent the total amount of sample that transferred to the membrane, total protein is measured on the membrane directly. This value is then used as the denominator when normalizing.1-4 Many total protein stains used to stain gels and membranes are commercially available.1 Total protein stains provide a larger dynamic range and demonstrate lower variability and cleaner data than housekeeping proteins.1,2

Total protein normalization can also be much faster than using a housekeeping protein, especially for chemiluminescent Western blots. This is because the time it takes to stain the blot takes less time compared to stripping and reprobing. Ideally, total protein staining is conducted on the membrane, either before or after immunodetection.2 Using some stains, such as AzureRed Fluorescent Total Protein Stain, it is possible to stain the blot before immunodetection and then to image total protein simultaneously with the protein(s) of interest. For Western blots and gels, AzureRed is able to detect less than 1 ng of protein per band or spot and is non-toxic and biodegradable, for safe and simple disposal.

AzureRed showed superior correlation and a much broader dynamic range than the common housekeeping proteins.
AzureRed showed superior correlation and a much broader dynamic range than the common housekeeping proteins, such as GAPDH.

AzureRed is a quantitative, fluorescent protein stain for total protein normalization in blots and total protein detection in gels. It is fully compatible with downstream Western blotting or mass spectrometry.

AzureRed Fluorescent Total Protein Stain
Choose AzureRed Fluorescent Protein Stain for sensitive detection of total protein on 1D or 2D gels. AzureRed is as sensitive as silver stain and is compatible with downstream Western blotting, mass spectrometry, and Edman sequencing. It provides very low background and higher signal to noise than other fluorescent stains and can be imaged with UV or blue light excitation.

AzureRed allows you to stain 1D and 2D gels in less than 3 hours, with high sensitivity, low background, and no speckling. Stained gels and blots can be imaged on both the new Sapphire FL (or other laser-based systems) and the Azure Imaging Systems (or other CCD-based fluorescent imaging systems).

Advantages and disadvantages with using Housekeeping Proteins for Normalization

BenefitsChallenges
Housekeeping protein• Familiar and commonly used• Narrow, linear dynamic range • Abundance can vary with experimental conditions • Abundance may not be consistent between sample types • High variability • Must ensure housekeeping protein physically resolves from protein of interest on the gel
Total protein• Larger linear dynamic range • Low variability • Constant across sample types • No change with experimental conditions• Must ensure the total protein stain that's used is compatible with antibody binding and detection method

Table 1. Benefits and challenges of using a housekeeping protein vs. total protein for Western blotting

Housekeeping Protein

BENEFITS
– Familiar, commonly used

CHALLENGES
– Narrow linear dynamic range
– Abundance can vary with experimental conditions
– Abundance may not be consistent between sample types
– High variability
– Must ensure housekeeping protein physically resolved from protein of interest on gel

Total Protein

BENEFITS
– Larger linear dynamic range
– Low variability
– Constant across sample types
– No change with experimental conditions

CHALLENGES
– Must ensure total protein stain used is compatible with antibody binding and detection method

Inconsistent levels

The most significant drawback of using housekeeping proteins is their levels may not be consistent across samples and conditions.1,2 While it is possible to use a housekeeping protein for normalization, but you must first spend the time and effort to validate your choice. You may also need to examine multiple potential standards before you find one that is truly expressed at the same level across all of your samples and does not change across your experimental conditions.

High abundance

A second significant challenge associated with housekeeping proteins is their high abundance.1,3 If the housekeeping protein is present at a very high level in your sample, this limits the amount of sample you can load on the gel because you will need to keep the housekeeping protein within the linear range of detection and not saturate the signal for the housekeeping protein. This is particularly problematic if the protein of interest is not similarly highly expressed, because the two proteins will not be within the same linear range of detection.2,3

Generating primary and secondary antibodies from non-overlapping species is difficult

A third challenge to consider if you’re doing multiplex Western blots, such as comparing phosphorylated and non-phosphorylated forms of the same protein, is the complexity of generating primary and secondary antibodies from non-overlapping species.

Keep in mind it is always possible that detecting the housekeeping protein could interfere with detection of the protein of interest.1 Ideally, the housekeeping protein should be a different size than the protein of interest, so the two proteins are spatially resolved on the Western blot. This becomes increasingly difficult when an experiment examines multiple proteins of interest on the same Western blot.

 

>> ADDITIONAL READING: Multiplex fluorescent Western blotting

Analysis after using a total protein stain

Comparison of a traditional western blot workflow to a western blot workflow using AzureRed Total Protein Stain
Comparison of a traditional Western blot workflow to a Western blot workflow using AzureRed Total Protein Stain

The analysis workflow after image capture is essentially unchanged compared to using a housekeeping protein; the signal density for the entire lane or a large portion of the lane is used for normalization instead of the density for a single band.

Staining the membrane with a total protein stain provides an added quality control benefit, allowing verification that membrane transfer was complete and free of artifacts. With this very simple workflow, images for the protein(s) of interest and total protein are automatically aligned, avoiding the need resize and align images captured at different times.

While you’re here, check out this brochure for a complete overview of available total protein stain options. For more on how to perform accurate Western blot normalization using AzureRed Fluorescent Protein Stain, check out this application note. Cheers for now.

Additional blog posts regarding total protein:

Frequently Asked Questions

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. Read more

TPN uses the entire protein content of each sample for normalization instead of relying on only a single housekeeping protein. You can see an example of total protein staining here.

AzureRed is a perfect choice for staining applications, including post-transfer staining to confirm uniform protein transfer from gel to membrane, and
staining quantitative Western blots as part of a TPN protocol. Read more

Azure offers a 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 TotalStain Q between protein transfer and blocking, and process them as you would normally. Read more

 
Shop Total Protein Stains

SOURCES

  1. Moritz CP. Tubulin or not tubulin: heading toward total protein staining as loading control in Western blots. Proteomics. 2017;17:1600189.
  2. Thacker JS et al. Total protein or high-abundance protein: which offers the best loading control for Western blotting? Anal Biochem. 2016;496:76-78.
  3. McDonough AA et al. Considerations when quantitating protein abundance by immunoblot. Am J Cell Physiol. 2015;308(6):C426-C433.
  4. Fosang AJ, Colbran RJ. Transparency is the key to quality. J Biol Chem. 2015;209(50):29692-29694.

Azure to Exhibit at 2023 AACR Annual Conference in Orlando, FL

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News Press Releases

Dublin, Calif. – April 3, 2023 ­– Azure Biosystems will be exhibiting at the 2023 Annual Meeting of the American Association for Cancer Research (AACR) in Orlando, FL at the Orange County Convention Center. This will be Azure’s first attendance at the event since 2019. The Azure team will be led by Tram Tran (Associate Marketing Manager), Vanna Sombatsaphay (Application Scientist, qPCR), and Justin Menko (Technical Product Specialist), who are excited to show meeting attendees Azure’s complete line of imaging and qPCR products. 

Azure Biosystems AACR 2023
In attendance for Azure at 2023 AACR will be Vanna Sombatsaphay (Application Scientist, qPCR), Justin Menko (Technical Product Specialist), and Tram Tran (Associate Marketing Manager).

Visit the team at booth #304 to see how easy gel imaging and densitometry can be with Azure’s brand-new personal chemiluminescence imager, the chemiSOLO. Also on display will be the new Sapphire FL Biomolecular Imager, a flexible imaging system with completely customizable and user-changeable optical modules. The Sapphire FL is compatible with nearly any imaging application, from gels and blots, to slides and small living animals, thanks to the addition of five anesthesia ports. Other flagship products from Azure that will be on display include the Cielo qPCR system, as well as the Azure Imaging Systems; all products will be available for in-person demos. The 2023 AACR Annual Meeting takes place April 14-19, 2023.

Is your AACR schedule quickly filling up? Schedule a meeting with someone from our team to make sure you don’t miss out! Schedule your meeting time here.

About Azure Biosystems

Azure Biosystems Inc. is an innovative life science platform company that designs, develops and markets state-of-the-art instruments, including the Azure Imaging Systems and the Azure Cielo Real-time PCR system. Azure Biosystems’ experienced team has developed 2nd and 3rd generation imaging systems for the life science market, thus applying their technical and market knowledge in creating innovative industry standard setting platforms.

Contact

Tram Tran
tram.tran@azurebiosystems.com
(925) 307-7127

Multicolor fluorescence imaging with Sapphire used to probe the structure of DNA recombinase

Categories
Fluorescence imaging

In a recent publication in Nature Communications, Caldwell et al used the multi-color fluorescence imaging capacity of the Azure Sapphire Biomolecular Imager to study the structure of DNA recombinase1.

The RecT family of recombinases contains over 1500 members. These enzymes bind to and catalyze the annealing of two complementary pieces of single-stranded DNA (ssDNA). No protein structures of RecT family members from bacteria or prophages have been solved. Some structures of RAD52, a potential human homolog of RecT, have been reported without DNA and with ssDNA, but not with two pieces of ssDNA which would represent an important intermediate of the annealing process.

Since the release of this interview, 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.

Solving the LiRecT Structure

Gel-shift assays imaged using fluorescent imaging on Azure Sapphire Biomolecular Imager
A portion of Figure 8a from Caldwell et al (2022). Structure of a RecT/Redβ family recombinase in complex with a duplex intermediate of DNA annealing. Gel-shift assays testing whether mutated forms of the LiRecT protein can bind to Cy3- and Cy5-labeled strands of ssDNA. For each mutant and the wild-type (WT) version of the protein, each strand was added individually (labeled in the figure as 3 for Cy3, 5 for Cy5) or sequentially (labeled 35). This portion of the figure shows that for some amino acids, double mutations interfered with DNA binding while single mutations did not. The gel was imaged on the Azure Sapphire Biomolecular Imager. Licensed under CC BY 4.0.

In this new work, Caldwell et al have solved the structure of LiRecT, a member of the RecT family from the prophage Listeria innocua, bound to a DNA duplex intermediate. The 3.4Å structure shows that multiple LiRecT subunits form a helical filament with an external groove that binds an extended and “un-wound” DNA duplex. The structure confirms that there is some structural similarity between the RecT family and the human protein RAD52. Though there is a great deal of difference between the structures, the authors identified a common core of structural similarity in the DNA-binding groove, supporting the hypothesis that the enzymes share a common underlying mechanism of protein-mediated DNA annealing.

Designed for flexible choice in detection chemistry and samples

The new Sapphire FL brings precise quantitation of nucleic acids and proteins. As the first system on the market of its kind to allow user-interchangeable filter modules, the Sapphire FL offers a broad range of excitation and emission wavelengths.
Learn More about the Sapphire FL
Scientist changing optical modules on the new Azure Sapphire FL
Pick the modules that support your research. Changing the optical modules on the new Sapphire FL is simple and easy. The unique mechanism makes selecting lasers to match your dyes finally possible. Easily swap lasers, filters, and/or entire optical modules in under two minutes to suit the needs of your experiment.

Can mutated proteins still bind to ssDNA?

To confirm the importance of specific amino acids that appeared to contact the DNA in their structure, the authors mutated 21 amino acid residues to see if changing them interfered with DNA binding. Gel-shift assays were used to see if the mutated proteins could still bind to ssDNA. RecT is known to bind weakly to individual strands of ssDNA and strongly to two complementary strands when they are added sequentially. Therefore, the authors used two complementary strands of ssDNA, one labeled with Cy3 and the other with Cy5, and conducted gel-shift assays in which the DNA strands were added individually or sequentially to the mutated enzymes. The results were imaged on the Sapphire, whose multi-color fluorescence capability allowed the researchers to detect the migration of each oligonucleotide on the same gel. The results generally supported the interactions suggested by the structure, though double mutations were often needed to completely disrupt DNA binding.

The work is an important contribution to understanding the mechanism of action of the RecT family and in what ways it resembles and differs from that of RAD52.

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.

Additional research using Azure Sapphire

SOURCE

  1. Caldwell BJ et al. Structure of a RecT/Redβ family recombinase in complex with a duplex intermediate of DNA annealing. Nat Commun. 2022;13(1):7855.

2023 Scientific Publication Requirements for Western Blots and Gels

Categories
Western Blotting

You’ve worked hard on your research, so when it’s time to submit your work for publication, don’t forget to check the publication requirements for Western blots for each journal. With over 3,000 publications worldwide featuring our products, you could say our users know a thing or two about publishing. The imagers and systems from Azure Biosystems deliver the quality journals are asking for, but image capture is only the beginning. When capturing images of gels and Western blots, it is imperative to record relevant information to ensure an image meets the requirements for journal publication.

Autoradiograph of an SDS-PAGE gel using phosphorimaging from Azure Sapphire
Figure 1. The above figure was published in the Journal of Cell Biology and shows an analysis of phosphorylation levels on wild-type and mutant Dam1 complexes. (A) Autoradiograph of an SDS-PAGE gel (8–14%). (B) Coomassie blue–stained SDS-PAGE gel (8–14%). Licensed under CC BY 4.0.
Phosphor Imaging with the Azure Sapphire in an Investigation of Chromosome Segregation

General publication requirements for Western blots and gels

Unfortunately, publication requirements for Western blots (Figure 1) and gels are not always uniform. Some journals recommend formats other journals will not accept, so always check the guidelines of the specific journal you plan to submit before preparing your draft for review! Knowing this, you can plan your experiments so you don’t need to cut apart the image of your gel or blot and reassemble it to create a figure. Avoid cutting and cropping your images more than necessary. Make sure internal controls are included on every gel or blot. Multiplex fluorescent Western blots are an excellent way to image your control with one or more targets on the same blot.

Some journals accept a variety of image types in the submission stage, but impose strict formatting rules once the manuscript is accepted. At the submission stage, some journals simply require figures be legible and provided in a format that is compatible with a broad range of operating systems and visualization programs. Others recommend figures be in the preferred publication format throughout all stages of the submission process.

Once a paper is accepted for publication, the requirements for Western blots and gels become much more specific and vary from journal to journal. In general the following guidelines will help ensure your image is publication ready:1

  • Capture images with at least 300 dpi and at least 190 mm wide.

    You can always shrink an image if it is too large, but you cannot increase the size of an image and many journals explicitly ban "upsampling." Using the Azure 600 Western blot imager makes capturing and saving large files easy.

  • Save a raw version of the image with no manipulations, including brightness and contrast.

  • Keep a record of the settings used to capture the image (resolution, exposure time, etc.)..

  • For modified versions of the image, keep a record of all manipulations that were performed to achieve that version of the image (brightness adjustments, channels overlaid, etc.).

Two scientists working on Azure 600
The Azure 600 uses a 9.1MP camera to provide high resolution imaging perfect for publications. Change the sample to optics distance using adjustable height shelf for enhanced detection. Zoom into the area of interest with ROI imaging to reduce background.
The Best Western Blotting Systems for Publication

Journal-specific publication requirements for Western blots and gels:

You should be aware of specific requirements for Western blots and gels when capturing images and preparing figures. Each journal will provide general guidelines for file types, file size, resolution, and color mode which apply to all images, including gels and Western blots. A list of general guidelines for publishing Western blots and gels in popular journals, such as Elsevier, Nature, Science, and Wiley are outlined below:

Best practices for preparing gel and Western blot images for publication

Creating figures from images of gels and Western blots (Figure 2) often presents a challenge. Authors must balance presenting the most relevant lanes and regions of a gel or blot with providing an accurate representation of the “big picture” of the experiment. Careful attention in the experimental design phase can help simplify this process. When planning the layout of the gel, think about how the data will be presented in an eventual figure and arrange samples in a logical manner. Do not include extraneous or unrelated samples in between the samples you plan to compare.

Western blots captured using Azure c600, published in nature
Figure 2. This figure was published in nature and shows Western blots captured using Azure c600 from Pascini et al. Immunoblotting evaluating the tissue-specific expression of PAI-1 in the transgenic mosquito.
Azure c600 Used in a Study to Research Malaria Transmission in Mosquitoes

Whenever possible, comparisons should only be made between samples run on the same gel. Internal controls, housekeeping protein standards, or total protein stains should always be processed on the same gel as the experimental samples. Therefore, in the planning phase, consider ways to optimize the amount of data to be generated from running just one gel. If one gel is not possible, make sure to include a control on each gel.

A recent publication by Kroon et al examined published Western blot figures and found a majority are cropped, missing essential information about the methods, and do not supply the original images as supplementary information. This reading provides recommendations to make Western blot figures more informative and reproducible, such as minimizing cropping and including (and labeling) molecular weight markers in all images.

Two scientists smiling holding Azure pub mugs
Are you getting ready to submit your paper for publication? Congratulations! We want to recognize your hard work. If you have published using an Azure Imager, post your publication on social media using the hashtag #ImagedbyAzure and tag us, we'll send you a pub mug to show off!

Editors at the Journal of Cell Biology published an article in 2004 addressing the temptation to alter or “beautify” images and describing acceptable and unacceptable manipulations of digital images. It provided an overview of the guidelines for blot and gel images that had been published to date by a variety of journals. Adjustments applied evenly across the entire image such as adjustments to brightness, contrast, or color balance are generally acceptable; however, it is always preferable to use an image that does not require such adjustments. For example, if your bands of interest in your Western blot are faint, it is better to take a longer exposure for publication rather than choose to digitally adjust the faint image to increase perceived band strength.

QUANTITATIVE WESTERN BLOT BASICS
quantitative western blot basics

CLAIM YOUR FREE QUANTITATIVE WESTERN BLOTTING BASICS GUIDE

Get a quick overview of the steps you can take to ensure your Western blots are quantitative. This free guide also includes a troubleshooting section and tear-out quantitative Western blotting checklist.

Download Guide

Nonlinear adjustments to an image should be avoided if you’re submitting to a journal. If they are used, carefully document the adjustments that were made. Some journals will require that these adjustments be described in the methods or figure legends. It is never acceptable to digitally alter the data in an image of a gel or blot; do not adjust contrast to hide background or faint bands. Those “nonspecific” bands may indicate your Western blotting conditions were not ideal and you need to change your blocking buffer or adjust your antibody concentrations. Alternatively, such bands maybe contain data whose importance will only become clear in the future. Maybe that “extra” band is actually an isoform of your protein, or a cleavage product.

Always save the original images used to make a figure. Some journals will request original images during the review process. Some journals, like the Nature portfolio journals in the life sciences, require original, unprocessed images of gels and Western blots used in figures be published as Supplementary Information (Figure 3).

Journals vary in how they prefer to receive figures in initial submissions. Always look up the specific requirements for the journal to which you are submitting, including required naming conventions for figure and image files. In summary, remember to capture high-resolution images (at least 300 dpi) and to carefully record your imaging settings. If you adjust an image, keep track of exactly what changes you made and always maintain a copy of the original raw image.

If you’re looking for an imager that’s reliable and provides sharp, clear images with every scan, look no further than a system from Azure Biosystems. Browse this list of available systems and choose the system that works best for your studies, like the Azure Sapphire Biomolecular Imager. The Sapphire is a next generation NIR fluorescent scanner equipped with lasers that delivers unmatched flexibility and performance for phosphor imaging, Western blots, animal imaging, in-cell Westerns, and more. Learn more about the Sapphire by clicking here.

More resources on Western blotting:

SOURCE
  1. Rattan, U.K.; Kumar, S.; Kumari, R.; Bharti, M.; Hallan, V. Homeobox 27, a Homeodomain Transcription Factor, Confers Tolerances to CMV by Associating with Cucumber Mosaic Virus 2b Protein. Pathogens 2022, 11, 788. https://doi.org/10.3390/ pathogens11070788

Imaging Coomassie-stained gels using NIR fluorescence and white light

Categories
Imaging SDS-PAGE

What is Coomassie?

Coomassie is a blue stain used in proteomics-related studies to detect proteins during electrophoresis, SDS-PAGE, and Bradford assays. In Western blot analysis, Coomassie is used as a loading control and an anionic pre-antibody stain. Coomassie binds non-specifically to almost all proteins. It is also the most common method of in-gel protein detection, whose popularity can be attributed to its characteristics of being efficient, quick, and affordable. 

In this blog post, we will cover how to use white light and NIR fluorescent to image coomassie-stained gels.

How to image Coomassie stained gels using white light or using NIR fluorescence

To begin, soak the gels in the dye. To reduce background and make the bands easier to visualize, elude the extra stain with a solvent; this step is called destaining. Destaining can take as little as 10 minutes, to as long as overnight to produce bands with clear background.

Doing these steps allows for the visualization of proteins as blue bands on a clear background. Below we show a serial dilution of bovine serum albumin (BSA) run on an SDS-PAGE gel and stained with a colloidal Coomassie stain. The same gel was imaged 3 ways.

Coomassie-stained gel imaged on the Azure 300 Imager on a white light transilluminator
Figure 1. Coomassie-stained gel imaged on the Azure 300 Imager on a white light transilluminator
Learn More about the Azure 300
Coomassie-stained gel imaged on the Azure chemiSOLO
Figure 2. Coomassie-stained gel imaged on chemiSOLO using white light
Learn More about chemiSOLO

The white light images were captured after placing the gel on a white light transilluminator table using white light on the Azure 300 Imager (Figure 1) and new chemiSOLO (Figure 2).

Coomassie-stained gel imaged in the 700 channel (NIR fluorescence) on the Azure Sapphire Biomolecular Imager
Figure 3. Coomassie-stained gel imaged in the 700 channel (NIR fluorescence) on the Sapphire Biomolecular Imager
Learn More about the Sapphire

The NIR fluorescence image was captured using the 700 channel (excitation wavelength 685nm) on the Sapphire Biomolecular Imager (Figure 3). On all images, the protein bands are easily visible down to the lowest amount loaded: 90 ng.

NIR fluorescence imaging of Coomassie-stained gels

About 15 years ago, it was reported that Coomassie blue-bound protein fluoresces in the near infrared3. Since that time, infrared fluorescent imaging of Coomassie stained gels has not become routine, perhaps limited by the availability of instruments able to carry out near-infrared fluorescence imaging3. Butt et al conducted a systematic study comparing the sensitivity and linear dynamic range of seven commercial Coomassie stains and seven published stain formulations when imaged using NIR fluorescence3.

The Coomassie stains were also compared to Sypro Ruby, which, though expensive, is a popular fluorescent stain due to its high sensitivity, high dynamic range, low interprotein variability, and mass spec compatibility. Butt et al found that some Coomassie stain formulations exceeded Sypro Ruby with respect to sensitivity and linear dynamic range3.

In examining 2D gels of mouse brain extract, Butt et al found more proteins were detected in gels stained with Sypro Ruby. The authors hypothesized the difference was due to attenuation of Sypro Ruby fluorescence in high-abundance spots, which may allow longer imaging to detect low abundance spots without signal saturation. This would suggest a tradeoff between sensitivity and quantitative accuracy when deciding between Sypro Ruby and Coomassie to stain a 2D gel3.

The sensitivity and linear dynamic range observed in this study suggests Coomassie may be an attractive, less expensive option for fluorescent staining of protein gels in some situations.

Scientist using pliers to place gel inside Azure chemiSOLO interface
The new chemiSOLO is used to image Coomassie-stained gels, chemiluminescent Western blots, and more.
Explore chemiSOLO

White light imaging of Coomassie-stained gels

In a Coomassie-stained gel, the protein bands appear blue in a clear background may be examined by eye on the bench or on a light box. For record keeping, the gel may be imaged photographed with white light. The sensitivity of traditional Coomassie staining depends on the specific protein being assessed. Detection limit per protein band is about 30 to 100 ng of protein and can approach 10 ng2, 3. The sensitivity of traditional Coomassie staining is about 10 ng2.

In addition to R-250 (the original Coomassie Brilliant Blue ) and G-250 (the demethylated derivative of Coomassie Brilliant Blue ), colloidal Coomassie stains are available in which the dye is present in micelles1. Colloidal stains result in lower background than traditional Coomassie stains because the micelles are too large to enter the polyacrylamide gel matrix1. Colloidal Coomassie blue staining can provide higher sensitivity, as low as 1 ng2. depending on the formulation3,4, 5.

Why Coomassie staining is so popular for SDS-PAGE gels

Coomassie blue is one of the most commonly used dyes for staining proteins in SDS-PAGE gels. A variety of Coomassie stain formulations can be purchased commercially or mixed in the lab. The staining protocol typically involves incubating the gel in staining solution until bands are visible, followed by destaining to remove background.

The entire staining process with Coomassie can take from a few hours to overnight. Destaining reduces sensitivity and is essential with traditional Coomassie staining because high background is common. Coomassie dyes bind protein through electrostatic and hydrophobic interactions1.

Advantages of using Coomassie

Advantages of Coomassie staining include simplicity and affordability. It is also compatibile with downstream mass spectrometry analysis. 

Disadvantages of using Coomassie

Disadvantages include low sensitivity compared to other staining methods. Staining intensity can depend in part on amino acid composition of the proteins, which complicates the use of Coomassie for relative quantitation of different proteins1.

Looking for better ways to visualize your proteins? Azure Biosystems offers a range of imagers capable of imaging Coomassie-stained gels under white light (epi or trans-illumination). The new Azure chemiSOLO is able to easily and quickly image chemiluminescent Western blots without additional software downloads. It’s the first personal Western blot imager of its kind on the market! Get a quote for chemiSOLO by clicking here.

Frequently Asked Questions about coomassie

Coomassie blue stain binds proteins non-covalently so it is compatible with downstream analyses such as mass spectrometry which is frequently used to identify the protein in a spot or band from a gel. Multiple formulations of Coomassie stain are commercially available and a variety of staining techniques have been published. Read more about Visible Gel Imaging here.

When used in acidic conditions, Coomassie will bind to the hydrophobic, basic residues in proteins. Its color will change from a dull copper, red/brown hue to an intense shade of blue.

There are a number of issues that can arise during a Western blot transfer.  Many times it can be resolved by adjusting the transfer time, temperature or simply remaking the buffers. More troubleshooting tips are available in this blog post: Trouble-free Transfers.

SOURCES

  1. Smejkal GB. The Coomassie chronicles: past, present and future perspectives in polyacrylamide gel staining. Expert Rev Proteomics. 2004;1(4):381-387.
  2. Butt RH and Coorssen JR. Coomassie Blue as a near-infrared fluorescent stain: a systematic comparison with Sypro Ruby for in-gel protein detection. Mol Cell Proteomics. 2013;12(12):3834-3850.
  3. Butt RH and Coorssen JR. Coomassie Blue as a near-infrared fluorescent stain: a systematic comparison with Sypro Ruby for in-gel protein detection. Mol Cell Proteomics. 2013;12(12):3834-3850.
  4. Neuhoff V, Arold N, Taube D, Ehrhardt W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis. 1988;9(6):255-262.
  5. Candiano G, Bruschi M, Musante L, et al. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis. 2004;25(9):1327-1333.

Western Blotting Reagents Roundup – November 2022

Categories
Reagent Roundup 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.

Featured Studies in this Reagent Roundup

The role of the epithelial to mesenchymal transition in cancer drug resistance and recurrence

Multicolor near-infrared Western blots and a combined chemiluminescence and NIR blot imaged using Azure 500 Western blot imager
Figure S2 from Ingruber J et al (2022). Interplay between partial EMT and Cisplatin resistance as the drivers for recurrence in HNSCC. Licensed under CC BY 4.0. Multicolor NIR Western blots (panels A,B,D,E) and a combined chemiluminescence and NIR blot (C) were imaged on an Azure C500 imager.

In recent work, Ingruber et al1 hypothesized that head and neck squamous cell carcinoma (HNSCC) cells are in a partial EMT state, able to switch towards epithelial or mesenchymal phenotypes depending on environmental stimuli, and that this switch contributes to their proliferation and resistance to Cisplatin therapy.

As part of this work, the authors carried out chemiluminescent and near-infrared (NIR) fluorescent Western blots to assess levels of EMT protein markers. The authors used Radiance Plus substrate for chemiluminescent Western blots, and fluorescent secondary antibodies for the near-infrared blots. Western blots were then imaged using an Azure c500 imager. The work found that a partial EMT-like pathway appears to contribute to Cisplatin resistance in the cell line used, and that overexpression of an epithelial marker sensitized cells to Cisplatin while reducing a pro-EMT transcription factor. The results suggest future avenues to research and treat drug-resistant cancers.

The epithelial to mesenchymal transition (EMT) is a reversible process in which epithelial cells undergo biochemical changes to adopt a mesenchymal cell phenotype with increased ability to migrate and increased resistance to apoptosis. The EMT can play a role in normal processes such as embryogenesis and wound healing, but also contributes to cancer metastasis and tumor cell migration.

DISCOVER: Azure 500 Imager

SHOP: TotalStain Q

Lipid peroxidation in sporadic Alzheimer's disease

Western blot imaged by fluorescence immunoblotting using Azure Sapphire Imager

In a recent publication, Ramsden et al2 propose a new hypothesis for the mechanism behind sporadic Alzheimer’s disease (AD) in which the initiating factor of AD is lipid peroxidation of the apolipoprotein E protein (ApoE) and of the ApoE receptor.  AzureRed total protein stain was used to detect total protein  before immunoblotting. The blots were blocked with Fluorescent Blot Blocking Buffer and imaged with the Azure Sapphire Biomolecular Imager (Figure 3C and Figure 3D).

The peroxidation is hypothesized to disrupt important processes required for memory formation and maintenance of structural integrity, initiating a cascade that leads to AD. The proposed mechanism differs from the amyloid cascade hypothesis and would have important implications for AD prevention and therapeutics if confirmed. Lipid peroxidation is proposed to occur at the ligand-receptor interface of ApoE and the ApoE receptor where there are amino acid residues predicted to be susceptible to peroxidation.

Because polyunsaturated lipids are transported by ApoE, the ApoE-ApoE receptor interface may create a microenvironment favorable to lipid peroxidation. The hypothesis accounts for several observations about AD including the anatomic areas of the brain known to be affected, the fact that ApoE variants are associated with sporadic AD, that ApoE is enriched in neurite plaque cores, the significance of amyloid plaques and neurofibrillary tangles, and evidence that lipid peroxidation occurs very early in sporadic AD. To test their hypothesis, the authors conducted fluorescence immunoblotting to detect lipid aldehyde-induced crosslinking of ApoE and the ApoE receptor ApoER2.

Based on these in vitro experiments and additional experiments including immunohistochemistry of human brain samples, the authors conclude that their hypothesis is consistent with experimental observations and deserves additional study.

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A study of the anti-inflammatory effects of LRP1 ligands

Mantuano et al4 used 3 ECL substrates (Radiance, Radiance Q, and Radiance Plus) from Azure Biosystems in their investigation of the anti-inflammatory action of three ligands of LDL receptor protein-1 (LRP1). Chemiluminescent Western blots imaged on the Azure c300 or on film were key to the study as the authors assessed the components required for enzymatically-inactive tissue-type plasminogen activator (El-tPA), activated α2-macroglobulin (a2M), and a soluble derivative of nonpathogenic cellular prion protein (S-PrP) to activate signal transduction in macrophages.

The results found indicate that lipid rafts and the N-methyl-D-aspartic acid (NMDA) receptor are required by all three ligands studied, while LRP1 was not required by two of the ligands when the ligands were present at high concentrations. In addition to the effects on cell signaling, the ligands studied were also shown to prevent lipopolysaccharide (LPS)-induced shedding of LRP1. Since the soluble LRP1 product is pro-inflammatory, blocking this process is another way LRP1 ligands could convey an anti-inflammatory effect. The differences uncovered between the three ligands’ requirements for signal transduction activation might help clarify their effects on macrophages in various states of differentiation.  

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SOURCES

  1. Ingruber J, et al. Interplay between partial EMT and Cisplatin resistance as the drivers for recurrence in HNSCC. Biomedicines. 2022;10(10):2482.
  2. Ramsden CE, et al. Lipid peroxidation induced ApoE receptor-ligand disruption as a unifying hypothesis underlying sporadic Alzheimer’s disease in humans. J Alzheimers Dis. 2022;87(3):1251-1290.
  3. Mantuano E et al. The LRP1/CD91 ligands, tissue-type plasminogen activator, a2-macroglobulin, and soluble cellular prion protein have distinct co-receptor requirements for activation of cell-signaling. Sci Rep. 2022;12(1):17594.
  4. Jäntti MA, et al. Palmitate and thapsigargin have contrasting effects on ER membrane lipid composition and ER proteostasis in neuronal cells. Biochim Biophys Acta Mol Cell Biol Lipids. 2022;1867(11):159219.

New tools created for the study of coagulation pathway proteases

Categories
Fluorescence imaging Imaging

In recent work, Modrzycka et al from Wrocław University of Science and Technology reported the development of novel activity-based probes (ABPs), inhibitors, and substrates specific to three serine proteases important to the blood coagulation pathway using data collected from an Azure Sapphire. The new reagents make it possible to differentiate these highly related proteases and study them simultaneously in complex biological samples. 

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.

Fluorescent ABPs and covalent inhibitors

To allow real-time detection of protease activity, the authors created ABPs labeled with fluorescent dyes (Cy3, Cy5, or Cy7) instead of biotin. The probes served as covalent inhibitors of the proteases. Versions of the covalent inhibitors without the fluorescent probes were also characterized. To demonstrate specificity in vitro, purified proteases were incubated with the fluorescently labeled ABPs, run on a gel, transferred to membrane, and fluorescence imaged using an Azure Sapphire Biomolecular Imager.

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Simultaneous detection of coagulation factors in human plasma

The authors then demonstrated that all three proteases could be detected individually in a complex biological sample using their specific fluorescent ABPs. Human plasma was incubated with each labeled probe and then separated on a gel. The gel was directly scanned on the Sapphire to detect each of the three fluorescent labels. As seen in figure 6C above, each of the three fluorescent probes is specific for its target protease. All three proteases could be detected simultaneously in the sample.

The authors conclude the newly developed tools may be used in multiple ways including the study of these proteases, pharmacologic knockdown of individual proteases, and diagnostics.

Parallel imaging of coagulation pathway proteases activated protein C, thrombin, and factor Xa in human plasma. Chem Sci. 2022;13(23):6813-6829. Licensed under CC BY-NC 3.0. This panel shows three fluorescently labeled activity-based probes, designed to be specific for the three coagulation factors APC, fXa, and thrombin (fiiia), detected in-gel bound to their respective targets in human plasma. Direct in-gel imaging was performed at 520 nm for Cy3, 658 nm for Cy5, and 784 nm for Cy7 with the Azure Biosystems Sapphire Biomolecular Imager
Figure C from Modrzycka et al. Figure FC. Parallel imaging of coagulation pathway proteases activated protein C, thrombin, and factor Xa in human plasma. Chem Sci. 2022;13(23):6813-6829. Licensed under CC BY-NC 3.0. This panel shows three fluorescently labeled activity-based probes, designed to be specific for the three coagulation factors APC, fXa, and thrombin (fiiia), detected in-gel bound to their respective targets in human plasma. Direct in-gel imaging was performed at 520 nm for Cy3, 658 nm for Cy5, and 784 nm for Cy7 with the Azure Biosystems Sapphire Biomolecular Imager.

Characterization of protease substrate specificity

Modrzycka et al used libraries of peptides including natural and non-natural amino acids to probe the substrate specificities of APC, thrombin, and factor Xa at each of four amino acid positions in the protease cleavage site. Peptide substrates were then designed based the results, incorporating amino acids at each of four sites to try to obtain substrates that were specific to each protease. The authors successfully identified substrates that were efficiently hydrolyzed by each protease while demonstrating little or no detectable hydrolysis by the other two proteases.

Activity-based probes to study coagulation factors

Until now, it has been difficult or impossible to differentiate between active and inactive forms of these proteases in situ. Antibodies are usually unable to distinguish between the zymogen and the active protease. Therefore, the authors created biotin-labeled versions of their specific substrate sequences. These activity-based probes can be used to track active versions of the proteases by SDS-PAGE, or to affinity purify the desired protease. The authors identified first-generation biotin-labeled peptides that retained potency and were highly specific for the target protease. To demonstrate specificity, enzymes incubated with each ABP were run on a gel, transferred to a membrane, and detected with fluorescently labeled streptavidin on the Sapphire.

The components and pathways that make up the classical blood coagulation cascade. Dr Graham Beards. Licensed under CC BY-SA 3.0
The components and pathways that make up the classical blood coagulation cascade. Dr Graham Beards. Licensed under CC BY-SA 3.0

The blood coagulation pathway

Blood coagulation involves a proteolytic signaling pathway in which a series of zymogens are activated by peptide bond cleavage to become proteases that in turn cleave additional targets. The pathway culminates in the cleavage of prothrombin to create thrombin, which promotes clot formation by activating platelets and creating fibrin from fibrinogen. Disruption of the blood coagulation pathway or abnormal activity of its players is associated with disorders ranging from hemophilia to cancer to Alzheimer’s disease. Therefore, understanding the pathway and how it is regulated is important to a variety of lines of research. 

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Challenges to studying regulation of the blood coagulation pathway

Activated protein C (APC), thrombin, and factor Xa, are proteases important to regulating the blood coagulation pathway and maintaining the correct balance of coagulation and blood flow. However, all three are vitamin K–dependent serine proteases and have similar structures and substrate preferences. The structural and substrate similarities have made it difficult to tease apart the roles of each of these three proteases in biological systems and disease states.

In addition to multicolor fluorescent imaging of blots and gels, the Sapphire Biomolecular Imager can carry out chemiluminescence, densitometry, white light, phosphor, and near infrared imaging of blots, gels, tissues, microwell plates, and more. Learn more about the Sapphire Biomolecular Imager and how Azure can support your research by clicking here.

 

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SOURCE
  1. Modrzycka S et al. Parallel imaging of coagulation pathway proteases activated protein C, thrombin, and factor Xa in human plasma. Chem Sci. 2022;13(23):6813-6829.

How is PCR used in genotyping?

Categories
qPCR

Polymerase chain reaction (PCR) is used in many genotyping approaches and methodologies. Simple PCR can be used for genotyping in situations where a known genetic sequence is being tracked, such as identifying model organisms carrying a transgene. PCR primers can be designed to only amplify a product when the animal’s DNA is carrying the specific sequence scientists have introduced. In these cases, the expected PCR product will be identified as a band on a gel.

Allele-specific PCR

Allele-specific PCR involves designing PCR primers that amplify different products depending on the genotype of the sample. Allele-specific PCR strategies can identify whether a sample is heterozygous or homozygous for a specific genetic variant based on whether one or two products are produced. Allele-specific PCR reactions can be analyzed by examining the sizes of the PCR end products on a gel or can be analyzed by following the accumulation of fluorescence using quantitative real-time PCR (qPCR).

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Using qPCR to detect SNPS

qPCR is frequently used to detect SNPs. With careful planning, PCR primers can be designed that are sensitive to the single base change at the location of the SNP and will only anneal to one variant so that a PCR product is only produced when the variant matches the primer exactly. In this type of system an intercalating fluorescent dye such as SYBR green can be included in the PCR reaction to follow the accumulation of the PCR product in real time.

Other strategies incorporating fluorescently tagged primers or probes have been developed that increase the specificity of the approach and allow multiplexing, the detection of two or more products in the same PCR reaction by qPCR.1

qPCR in genotyping
Figure 1. View of the Cielo's Experiment Area screen showing the increase over time of the fluorescent signals reflecting PCR product accumulation in a multiplex qPCR reaction
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Other ways to detect SNPS

Different PCR strategies can also be incorporated into genotyping approaches that can be used even if you don’t know anything about the genome of the organism you are studying. In random amplified polymorphic DNA (RAPD), random 10-mer primers are used to carry out PCR, and the products generated across different samples are compared, usually by separating them on a gel and comparing the sizes of the amplified products.

For the RAPD approach to work, the combination of primers must produce products and in some fraction on individuals, genetic variation changes the size or number of PCR products. With luck, some of the genetic differences reflected in the different PCR products will associate with the phenotype of interest.2

Using amplified fragment length polymorphism to genotype

Another genotyping strategy that incorporates PCR and does not require knowing the sequence of the genome of the organism being studied is amplified fragment length polymorphism (AFLP). In AFLP, the genomic DNA is digested with restriction enzymes, adapters are ligated to the digested ends, PCR is carried out using primers directed towards the adapters, and the products are separated and analyzed by gel electrophoresis. As in RAPD, the method is useful if there are some differences in PCR products that are associated with the phenotype of interest.3

More blog posts to read about qPCR:

SOURCES

  1. Broccanello C et al. Comparison of three PCR-based assays for SNP genotyping in plants. Plant Methods. 2018;14:28.

  2. Random Amplified Polymorphic DNA (RAPD) at the National Library of Medicine website. https://www.ncbi.nlm.nih.gov/probe/docs/techrapd/. Accessed October 31, 2022.

  3. Paun O, Schönswetter P. Amplified Fragment Length Polymorphism (AFLP) – an invaluable fingerprinting technique for genomic, transcriptomic and epigenetic studies. Methods Mol Biol. 2012; 862:75-87.

Sapphire Biomolecular Imager used in investigation of potential nasal vaccine for COVID-19

Categories
COVID-19 Imaging

Vaccines have been instrumental in the public health response to the SARS-CoV-2 pandemic. The existing approved vaccines induce neutralizing antibodies and are very effective at preventing symptomatic infection. However, current vaccines do not elicit sterilizing and mucosal immunity necessary to avoid breakthrough infections.

Testing an attenuated version of SARS-CoV-2

In a recent publication, Ye et al (2) set out to make an attenuated version of SARS-CoV-2 and test its utility as a vaccine. The authors created a recombinant strain with a point mutation that inactivates NSP16, a protein needed to cap viral RNAs and prevent them from being detected by the immune system. The mutation, D130A, was known to inactive the analogous enzyme in other coronaviruses. The mutant SARS-CoV-2 strain was characterized to determine if (1) the virus was viable, (2) the ability of the virus to replicate was attenuated, and (3) the virus could be a candidate for vaccine development.

Fig. S1. Genetic stability of SARS-CoV-2 d16. A Detection of d16 gene during viral passage in VeroE6 cells. RNAs were extracted from the d16-infected cells of P0 to P10 passages. RT-PCR was performed with a primer pair flanking the d16 mutation. The 297-bp PCR products were resolved by agarose gel electrophoresis (arrowhead). The passage numbers were denoted at the top of each lane. B Sanger sequencing.

The findings

The answer to all three questions was “yes”. The virus was viable in in vitro assays, but its growth was attenuated both in vitro and in vivo. Hamsters infected with the mutant virus had significantly lower viral loads in the upper and lower respiratory tracts than hamsters infected with the wildtype virus, and infection with the attenuated virus did not kill transgenic mice as wildtype virus did.

Use of the mutant strain as a live vaccine was tested in hamsters using a single dose regimen. Hamsters were inoculated intranasally with wildtype or mutant virus, monitored for 28 days, then challenged with wildtype virus on day 29. On day 28, the presence of neutralizing antibodies was assessed using a live virus neutralization assay that detected bound virus using a fluorescently-labeled antibody. The fluorescent signal was assessed and quantified using the Azure Sapphire Biomolecular Imager.

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.

The new Sapphire FL is the ultimate biomolecular imager for flexibility. With customizable and user-changeable laser and filter modules, the Sapphire FL easily adapts to a lab’s changing needs and advancing research.
Learn More about the Sapphire FL

Exposure to the mutant strain was found to be as effective as the wildtype virus at preventing SARS-CoV-2 infection. Viral titers in the lung and respiratory tract were so low the vaccination may have elicited sterilizing immunity. Indeed, hamsters co-housed with infected but vaccinated hamsters never became infected themselves. Promisingly, spike protein-specific IgA was detected in the serum and bronchoalveolar lavage fluid of vaccinated mice, suggesting mucosal immunity was stimulated.

Ye et al conclude the results provide strong justification to pursue further developing the strain as a potential vaccine.

This illustration, created at the Centers for Disease Control and Prevention (CDC), reveals ultrastructural morphology exhibited by coronaviruses. Photo credit: Alissa Eckert, MSMI; Dan Higgins, MAMS.
This illustration, created at the Centers for Disease Control and Prevention (CDC), reveals ultrastructural morphology exhibited by coronaviruses. Photo credit: Alissa Eckert, MSMI; Dan Higgins, MAMS.

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How COVID infections begin

COVID infections usually begin in the upper respiratory tract mucosa. Therefore, stimulating mucosal immunity may be most effective at preventing infection. Mucosal immunity is independent of the systemic immune system and is mediated primarily by secretory IgA antibodies. Injectable vaccines such as those used for flu and pneumonia primarily induce serum IgG, which may protect the lower respiratory tract and prevent systemic spread. Inducing upper airway mucosal protection may require nasal immunization. Additionally, in some experiments inducing a mucosal cellular immune response has required immunizing with whole, wild-type or attenuated viruses or bacteria.(1)

Live attenuated vaccines use a weakened form of the infectious agent (virus or bacterium). The agent can replicate enough to stimulate a strong immune response but not so much as to cause disease. Live attenuated vaccines result in a strong and long-lasting immune response, are used to protect against several agents, and were crucial to the eradication of smallpox and the near eradication of poliovirus.

In addition to fluorescent imaging of tissue culture plates, the Sapphire provides densitometry, phosphor, multichannel fluorescence, near-infrared, chemiluminescence, 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. 

More research done with the Azure Sapphire:

SOURCES

  1. Holmgren J, Czerkinsky C. Mucosal immunity and vaccines. Nature Med. 2005;11:S45-S53.
  2. Ye ZW, Ong CP, Tang K, et al. Intranasal administration of a single dose of a candidate live attenuated vaccine derived from an NSP16-deficient SARS-CoV-2 strain confers sterilizing immunity in animals. Cell Mol Immunol. 2022;Mar 29;1-14.
  3.  

In-cell Westerns used to Demonstrate Potent Anti-inflammatory Activity of Mosquito Saliva Protein

Categories
Imaging In-cell Western

Both the Azure 300 and Sapphire Biomolecular Imager, along with fluorescent blocking buffer and secondary antibodies from Azure Biosystems, were used in a recent publication revealing anti-inflammatory activity of a mosquito saliva protein. Shrivastava et al from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health (NIH) in Rockville, MD, employed both instruments in their study demonstrating how protein Alboserpin inhibits pro-inflammatory activities of the coagulation cascade protein factor Xa (FXa).

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.

Research Applications with the Azure 300 and Sapphire Biomolecular Imager

By traditional and in-cell Western blots, Alboserpin was shown to block the FXa-induced increase in phosphorylated ERK1/2, indicating it blocks FXa-induced ERK1/2 signaling. The chemiluminescent blots were imaged using the Azure 300, while the in-cell Western blots (Figure 1D) were imaged on the Sapphire with fluorescent blocking buffer and fluorescent secondary antibodies.

Figure 1D from Shrivastava et al (2022), Alboserpin, the main salivary anticoagulant from the disease vector Aedes albopictus, displays anti-FXa-PAR signaling in vitro and in vivo
Figure 1D from Shrivastava et al (2022), Alboserpin, the main salivary anticoagulant from the disease vector Aedes albopictus, displays anti-FXa-PAR signaling in vitro and in vivo. Licensed under CC BY 4.0. In-Cell Western blots conducted using Azure fluorescent secondary antibodies and imaged on the Azure Sapphire Biomolecular Imager demonstrate a decrease in Fxa-induced phosphorylation of EKR 1/2 in the presence of Alboserpin.

These results from the NIH provide additional insight into the mechanisms mosquito vectors employ to avoid host responses during blood feeding. The authors indicate further research is needed to examine the role Alboserpin might play in pathogen transmission.

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Alboserpin and its role on FXa

FXa is known to trigger inflammation by signaling through protease-activated receptors (PARs). In their recently published work, Shrivastava et al demonstrate that Alboserpin has anti-inflammatory activity, in vitro and in vivo, due to its inhibition of FXa activities. Across multiple experiments, Alboserpin was shown to prevent numerous FXa-induced processes including FXa-induced release of proinflammatory cytokines, increased expression of multiple PARs and other proteins (specifically PAR-1, PAR-2, PAR-3, NF-kB, ICAM, and VCAM-1), induced activation of NF-kB, and cleavage of PAR-2.

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What are Aedes mosquitos? Why is their saliva important?

Aedes mosquitos are an important vector for arthropod-borne (arbo) viruses such as Zika, dengue, and chikungunya viruses. Arbo viruses are responsible for substantial disease burden, morbidity, and mortality among human populations world-wide. These viruses can be introduced into a host when the mosquito pierces the skin to draw blood and releases saliva at the site of the bite.

The mosquito saliva contains proteins that prevent hemostasis (a process that involves reduced blood flow), coagulation, and clot formation to prevent blood loss at the site of an injury. The Aedes albopictus mosquito has an anticoagulant called Alboserpin in its saliva. Alboserpin is a serine protease inhibitor known to demonstrate competitive, reversible, and high-affinity binding to FXa, an important component of the coagulation cascade.

More about in-cell Westerns: History behind In-cell Westerns

In addition to chemiluminescent and multicolor fluorescent imaging of blots and multi-well plates, the Sapphire Biomolecular Imager can carry out densitometry, white light, phosphor, and near infrared imaging of blots, gels, tissues, and more. Learn more about the Sapphire Imager and how Azure can support your research by clicking here.

More published research using the Azure Sapphire:

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SOURCE

  1. Shrivastava G, Valenzuela-Leon PC, Chagas AC, et al. Alboserpin, the main salivary anticoagulant from the disease vector Aedes albopictus, displays anti-FXa-PAR signaling in vitro and in vivo. ImmunoHorizons. 20022;6(6):373-383.