When to use Wet, Semi-Dry and Dry Transfers for Western Blots

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Transfers Western Blotting

Choosing the right transfer method can determine your Western blot’s success. The first step to Western blotting is separating the proteins in a sample by size, using a denaturing process called gel electrophoresis.

How does Western blot transfer work?

In Western blotting, after the electrophoresis step, is the transfer step. The transfer step is important to Western blotting because it moves the separated proteins from the gel onto a solid support matrix using a membrane (either nitrocellulose or polyvinylidene difluoride (PVDF)). This is where the blot will form.

Cases where you would not need to transfer the gel

Sometimes, protein separation is not required, so your sample may be directly added to the membrane using an approach called “dot blotting.”

Three types of Western blot transfers

Your choice of transfer comes down to whether you need quantitative information from your blot, time and cost, or if your protein is finicky and requires you to customize transfer conditions (Table 1). So, wet or dry – which is the best transfer method for your Western blot? Let’s go over when to use each transfer method to best prepare you for your next Western blot procedure.

Table of contents

Table 1. When to know when to choose which Western blot transfer method

ScenerioWhich transfer method to use
You want to gain quantitative information from your Western blotWet transfer
Wet transfers allow you to customize the time, temperature, voltage, or buffer to best suit your protein of interest.
Saving time and reagents is equally importantSemi-dry transfer
In a semi-dry transfer, the only buffer used is the one that saturates the stack components (Figure 2).
You're short on timeDry transfer
Dry transfers typically only take around ten minutes to finish. They are also the least quantitative Western blot method.

How wet transfers for Western blotting work

If you want to gain quantitative information from your Western blot, you should do a wet transfer using a tank. Wet transfers are performed in a tank filled with transfer buffer (Figure 1). Most transfer systems, like the Azure Aqua Transfer Tank, have room for two precast or handcast gels. Tanks are routinely used in wet labs for Western blot transfers.

Azure Aqua during Western blotting experiment
The Azure Aqua Transfer Cell is used for transferring two mini gels to membranes for Western blotting experiments (wet transfer). The transfer cassettes and electrode core are colored for directionality which makes transferring easy and straightforward. You can perform a quick 1-hour transfer or you can transfer overnight at a lower voltage.

The transfer buffer used for wet transfer protocols is traditionally a Tris-glycine buffer, which contains methanol, but you may also use other buffers too. In certain cases, SDS may be added to the buffer to aid the transfer of large proteins. 

Wet transfer setup for electrophoresis
Figure 1. Wet transfer setup. A “stack” is built in which the gel is placed next to a membrane (nitrocellulose or PVDF), both of which have been pre-equilibrated in transfer buffer. Blotting papers and sponge, which have also been pre-soaked in transfer buffer, are added to the outside of the stack so the stack can be held firmly within a cassette that is suspended in the transfer buffer–filled tank. Electrodes on the cassette allow an electric current to be run through the stack so the proteins migrate from the gel to the membrane.
  • Quick Tip: The Transfer buffer from Azure Biosystems is specially is formulated to increase protein transfer and protein retention on the membrane for optimal sensitivity.

Pros:

  • Wet transfers are highly customizable. The time, temperature, voltage, and buffer can be varied to suit the protein of interest and to achieve complete transfer of a broad range of proteins.
    • For example, longer transfer times may be used to allow larger proteins to fully migrate out of the gel, while shorter transfer times can prevent loss of low-molecular weight proteins that may otherwise migrate through the membrane entirely.
    • The voltage can also be reduced to slow the transfer process for an overnight run, or increased to complete the transfer in an hour or two.
  • Overall less expensive than other transfer methods
  • Can use multiple buffers to optimize transfers
  • Transfers broad molecular weight range at one time
  • Extended transfer is possible
  • Can be used for quantitative Westerns

Cons:

  • One disadvantage of wet transfers is that heat is generated during the transfer. This can contribute to inconsistent transfers and to breakdown of the gel. To combat the effects of excess heat, wet transfers are often conducted in a cold room and/or with ice packs or cooling units in the tank.
  • Another disadvantage to wet transfers is they require a large volume of transfer buffer. If you regularly conduct a large number of transfers, reagent consumption can become an issue. Because of the larger volume of usage, you will also accumulate more hazardous waste. To conserve reagents, semi-dry transfer methods can be used.
  • Wet transfer can also take up to one hour. With some systems, you can also run transfers overnight at a low voltage.
  • Cooling mechanism and/or cold room space is required during transfer.

How semi-dry Western blot transfer methods work

In a semi-dry transfer, the only buffer used is the one that saturates the stack components. The membranes and gels will be placed between wet filter papers (a sandwich) that will be in direct contact with the electrodes (Figure 2). Semi-dry transfers should be your choice if saving time and reagents is your first priority.

Semi-dry transfer stack for electrophoresis
Figure 2. Semi-dry transfer setup. In a semi-dry transfer, the stack consists of gel and membrane placed between two pieces of filter paper, all equilibrated in transfer buffer. This stack is placed directly between two electrode plates.
  • Quick Tip: Transfer times cannot be extended for proteins that do not transfer completely with the standard protocol.

    The stack can dry out and the buffer capacity of the small amount of transfer buffer will be exhausted if transfer times are too long.

Pros:

  • Overall easy to setup. Good for performing large numbers of blots where you are analyzing the same protein.
  • With semi-dry transfers, transfer times are reduced to about an hour, but may be as short as five  minutes with rapid semi-dry transfer protocols.
  • The hazardous waste from transfer buffer is minimized because only small volumes are used each time.

Cons:

Semi-dry transfers run into difficulties at extreme ends of the protein size range. Large proteins may not transfer out of the gel quantitatively in the short transfer time available, while small proteins may transfer entirely through the membrane.

  • To compensate, you can use discontinuous buffer systems. With these systems, the two pieces of filter paper on either side of the stack are equilibrated in different buffers. For example, buffers can be chosen to help transfer difficult proteins out of the gel, and/or to improve retention of proteins in the membrane.
  • High intensity field strength may cause low molecular weight proteins to migrate through membrane.
  • Difficulty in transferring high (>120 kDa) molecular weight proteins
  • Not recommended for quantitative Western blots
  • Quick Tip: Even though discontinuous buffer systems can help improve transfer across a larger range of protein sizes, semi dry transfers are not recommended for quantitative Western blotting.

How “dry” Western blot transfer methods work

With dry transfer, the gel is placed between the preassembled stacks, that already contain the transfer membrane and proprietary buffer matrices you’ll need. Because of this convenience, if you are short on time, dry transfers are the best option.

Pros:

  • Dry Western blot transfer systems do not use transfer buffer at all, which saves time.
  • Can be completed in as little as ten minutes when using newer models.

Cons:

  • There is no opportunity to customize or optimize your solutions based on your protein of interest.
  • With dry transfers, preassembled stacks must be purchased ahead of time. This adds to reagent and consumable costs.

Remember: Western blot transfers are customizable!

Wet Western blot transfers are highly customizable and are recommended for quantitative Western blotting but consume a lot of reagents. Semi-dry Western blot transfers conserve time and reagents, but may not allow quantitative transfer for all proteins, especially those that are very small or very large. Dry Western blot transfers are the fastest of all and require no buffer preparation, but do not allow much room for optimization.

Looking for clear and consistent imaging results? Your search ends here

The Azure 600 uses a 9.1MP camera to provide high resolution imaging perfect for publications. Request a free demo of an Azure Imaging System, and say "Hello" to beautiful Western blots.
Two scientists working on Azure 600

In summary, the transfer method you choose will depend on several factors of your Western blotting experiment. One of the factors is how you balance the importance of speed and/or reducing reagent consumption vs needing quantitative transfers or to customize the protocol for a difficult protein. Check out the resources below if you’re still having trouble, or send us a message using the form on this page. Cheers for now!

Frequently Asked Questions about Western blot transfers

The reason for blocking the membrane before and during incubation of the primary antibody is due to the membranes being “sticky” to any protein. We “block” in order to cover up all parts of the membrane that don’t have protein on them, including any region in between lanes, etc.  When we add the antibodies, they will only bind to the protein of interest, not the blank membrane.  Blocking ensures we don’t get signal from primary antibodies sticking to the membrane. We will only get signal from primary antibodies binding to their protein of interest.

Want to try a new blocking buffer? We offer free samples!

The blocking agent may be protein or non-protein based. Some of the more commonly used blocking agents include: normal serum, Bovine serum albumin, non-fat, dry milk, Polyvinylpyrrolidone, or Tween 20. Read more about blocking agents in this application note.

There’s an application note for that! Check out our app note “Wet or Dry?” for a variety of buffer recipes.

Shop Western blotting accessories for electrophoresis

Common Western Blotting Questions, Answered

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Fluorescence imaging Quantification Troubleshooting Western Blotting

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

Answers to common Western blotting questions

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

ExploreWestern Blot Imaging Systems

What reagents do you need for Western bloting?

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

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

Why are black dots showing on my Western blot?

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

What is chemiluminescent detection?

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

Depiction of chemiluminescent Western blot signal
Chemiluminescent Western blotting- one signal, one protein. In chemiluminescent detection, the antigen-primary antibody complex is bound by a secondary antibody conjugated to an enzyme, such as horseradish peroxidase (HRP). The enzyme catalyzes a reaction that generates light in the presence of a luminescent substrate, and the light can be detected either by exposure to x-ray film or by a CCD-based imaging system.

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

What's more sensitive: chemiluminescence or fluorescence?

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

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

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

Continue readingBeginning Chemiluminescent Western Blotting

Shop chemiluminescent substratesRadiance Q

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

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

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

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

What is a chemiluminescent substrate?

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

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

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

Is HRP a chemiluminescent substrate?

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

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

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

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

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

Additional Western Blotting Resources

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!

Multiplex Western Blots: 6 Strategies that Work

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Multiplex Western Blotting

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 that cover phosphorylation/total protein detection, and single step normalization.

Detecting one protein, two proteins, three proteins? How about FOUR proteins on the same Western Blot? Multiplexing your Western blots allows you to save money on each experiment.

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.

Azure fluorescence capabilities vs. competitor fluorescence, 3 proteins, plus total protein staining, and multiplex fluorescent Western blot

Browse our 6 multiplex Western blot expert tips

AZURE EXPERT TIP #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.

AZURE EXPERT TIP #2: Test individually first

Going hand in hand with our first tip, 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 try to unpick 3 different primary and secondary antibodies at once.

AZURE EXPERT TIP #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.

Multiplex fluorescent Western blot
Multiplex fluorescent Western blot imaged with an Azure Imager using Cy3 and Cy5.

AZURE EXPERT TIP #4: Expand the spectrum with multiplexing

Three-color Western blots are exciting, but what about five colors? With near-infrared 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.

The Sapphire Biomolecular Imager is an imager capable of detecting up to four proteins on the same Western blot, which allows you to save money on reagents each time you do an experiment. It uses 4 fluorescence channels to easily quantify overlapping bands. Read more on multiplex protein detection here

Since the release of this blog post, 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.

AZURE EXPERT TIP #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.

Package of pre-cut PVDF membranes for Western blotting applications
PVDF membranes reduce background noise for improved sensitivity.

AZURE EXPERT TIP #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.

Want to read more? Check out this paper from the National Institute of Health that utilizes multiplex Western blots using microchip electrophoresis.

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.

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!

The Key to Early Alzheimer’s Detection

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Western Blotting

Alzheimer’s disease is the most common cause of dementia and affects over 6 million Americans.1  It is important to research early indications of Alzheimer’s. This age-related, progressive disease is marked by the accumulation of protein aggregates in the brain (beta amyloid plaques around neurons and tau tangles within neurons) and by a decrease in brain glucose metabolism. Surprisingly, studies with subjects who were genetically predisposed to Alzheimer’s disease have found that those two neurological symptoms can begin to occur decades before Alzheimer’s symptoms become apparent. Currently, it is not possible to identify Alzheimer’s disease during this long preclinical period when interventions might be most effective.

Identifying biomarkers that may serve as early indicators of Alzheimer’s disease is an important subject of current Alzheimer’s research. The ideal identifying biomarker would be one that could be measured using inexpensive and noninvasive methods. To this end, Yao et al investigated in a recent paper whether mitochondrial dysfunction could be detected in extracellular vesicles (EVs) from the blood of Alzheimer’s patients. Given that EVs are small vesicles released into biological fluids like blood from many types of cells, they have been shown to contain analyzable components from the cells that release them such as nucleic acids and proteins.

Figure 1 from Yao et al. (2021) Mitochondrial electron transport chain protein abnormalities detected in plasma extracellular vesicles in Alzheimer’s Disease. The Azure Biomolecular Imager was used to image chemiluminescent blots analyzing markers present in the extracellular vesicle prep (panel B). Licensed under CC BY 4.0.

The authors collected EVs from the plasma of patients with high-probability early Alzheimer’s disease and from control patients with no evidence of dementia. A neuronal marker was used to selectively enrich for EVs from neuronal cells. The resulting EVs were characterized by Western blot to demonstrate that they contained EV markers and not markers associated with potential contaminants. To maximize image clarity and ensure accuracy, the chemiluminescent Western blots were imaged using the Azure Sapphire Biomolecular Imager.

Biochemical assays revealed that neuronally derived EVs from patients with Alzheimer’s disease had significantly lower levels of mitochondrial electron transport chain (ETC) complexes and lower levels of superoxide dismutase than EVs from control patients. In addition, the EVs from Alzheimer’s patients had significantly reduced activity of ETC complexes IV and V. The authors concluded that the neuron-derived EVs demonstrate the same abnormalities observed in Alzheimer’s tissues and model systems of Alzheimer’s disease and therefore may provide a biomarker for detecting early mitochondrial dysfunction in Alzheimer’s disease.

In addition to chemiluminescence imaging, 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 and how Azure can support your research by clicking here.

 

SOURCES

  1. Yao PJ, Eren E, Goetzl EJ, Kapogiannis D. Mitochondrial electron transport chain protein abnormalities detected in plasma extracellular vesicles in Alzheimer’s Disease. Biomedicines. 2021;9(11):1587.
  2. Alzheimer’s Association. 2021 Alzheimer’s Disease Facts and Figures. Alzheimers Dement 2021;17(3).
  3. Mustapic M, Eitan E, Werner JK, et al. Plasma extracellular vesicles enriched for neuronal origin: A potential window into brain pathologic processes. Front Neurosci. 2017;11:278.

History Behind In-cell Westerns

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In-cell Western Western Blotting

Western blotting vs. In-cell Western blotting

Western blotting was first described by three groups in 1979. Since then numerous refinements to the basic technique, reagents used and imaging technologies have massively broadened the usage of Western blotting, making it a cornerstone protocol in life sciences today.

However, the general workflow of a Western blot has remained the same throughout this time. Samples are isolated and protein is extracted and standardized. Proteins are then separated by gel electrophoresis, transferred and probed with primary and secondary antibodies before being visualized. With numerous steps, Western blotting as a protocol requires a large investment in equipment, training, reagents and, perhaps most importantly, time.

standard Western blot protocol takes two days to work from sample to image. Therefore, when analyzing a large number of samples or performing optimizations, it is easy to see how Western Blotting can quickly become a bottleneck in your research workflow.

in-cell Western with Azure Sapphire laser scanner
A serial dilution of HeLa cells were seeded into a 96-well plate, cultured, fixed and permeabilized. The individual channels were scanned simultaneously then combined into a single composite image using the Sapphire Capture Software.

What is In-cell Western blotting?

Enter in-cell Western blotting – a radical rethinking of the standard protocol coupling the ability to accurately quantify intracellular proteins from Western blotting with the repeatability, quick turnaround and high throughput of an ELISA. Cells are grown on sterile plates, before being fixed and permeabilized in situ. Subsequent labelling is performed as normal with a specific primary antibody followed by an isotype specific fluorescent conjugated secondary antibody. Conjugates in the near-infrared (NIR) spectrum are commonly used to reduce auto-fluorescence and noise typically associated with tissue culture plastics.

The Azure Biosystems Sapphire™ FL Biomolecular Scanner is specifically designed to allow for visualization in two NIR channels (658 nm and 785 nm) as well as visible fluorescent wavelengths (488 nm and 520 nm), allowing for multiplex imaging within wells. This multiplex capacity allows for the assessment of multiple proteins of interest in a single well which is useful for looking at total and phosphorylated versions of a protein or to detect an in-well standard to allow for quantification across a plate.

Azure Sapphire FL Biomolecular Imager with lid open
With a laser-based excitation system using photomultiplier tube (PMT) and avalanche photodiode (APD) detectors, signal selectivity, sensitivity and speed of imaging achievable by the Sapphire FL make it an ideal choice for in-cell Western blotting.

In the application note below, we demonstrate the ability to quantify intracellular proteins in-situ using the Azure Sapphire. 

Since the release of this blog post, 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.

Quantifying Intracellular Proteins In-situ App Note

Materials and Methods

Culture cells

HeLa cells were serially diluted and seeded into a sterile 96- well tissue culture plate at a volume of 0.2 mL per well, and grown until approximately 80% confluent. All wells were fixed and permeabilized using 100% methanol for 15 minutes at room temperature.

In-cell Western blotting

Following fixation and permeabilization, cells were rinsed in PBS, blocked with 1% fish gelatin in PBS for one hour at room temperature, then probed for alpha-tubulin and beta-actin overnight at 4°C. Samples were washed three times with PBS prior to incubation with AzureSpectra 550 and AzureSpectra 800 conjugated secondary antibodies for 60 minutes.

Secondary antibodies were incubated along with RedDot™1 Nuclear Stain to normalize for total cell number. Plates were washed as previously described before imaging.

Imaging

After washing, the plate was imaged with the Sapphire using the Plate Focus Setting.

In- cell western performed using Azure Sapphire
Figure 2. A serial dilution of HeLa cells were seeded into a 96-well plate, cultured, fixed and permeabilized. A) Columns 1-3 were probed for beta-Actin using AzureSpectra 550 (green). B) Columns 3-6 were probed for Tubulin using AzureSpectra 800 (blue). C) The entire plate was stained with RedDot1 Nuclear Stain as a normalization control (red). D) The individual channels were scanned simultaneously then combined into a single composite image using the Sapphire Capture Software.

Results and Conclusions

HeLa cells were grown in a 96-well plate probed with alpha-tubulin and beta-actin antibodies followed by isotype appropriate secondary antibodies conjugated with AzureSpectra 550 and AzureSpectra 800 respectively. All wells were incubated with RedDot™1 Nuclear Stain to normalize for total cell number. Images were acquired using a Sapphire™ Biomolecular Scanner and are displayed in Figure 2. The representative image shown demonstrates the high level of sensitivity and specificity achievable.

Note: The above is an abbreviated protocol. You can find the full protocol here.

North Carolina’s Elite Christmas Tree Industry

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Customer Spotlight Imaging Western Blotting

Customer Spotlight: Adarsha Devihalli, PhD Candidate at North Carolina State

Since the release of this interview, the Azure c400 has been upgraded to the Azure 400, a flexible fluorescent imager that enables three color fluorescent detection for dyes in the visible range.

Nestled in the southern region of the Appalachian Mountains is an environmentally beneficial abundance of Fraser fir—the most sought-after Christmas tree in the USA. Thanks to its charming aroma, soft and durable needles, and eye-catching silhouette the tree forms the foundation of a multi-million dollar industry in North Carolina. It is these qualities combined with this unique geography that make North Carolina the second-leading Christmas tree producer in the United States. And while Fraser firs are heavily popular with holiday enthusiasts, they’re also extremely vulnerable to Phytophthora, a common cause of root rot disease.

"The Azure 400 Imager comes in [and is] a multi-user instrument…so we don’t have to run different instruments or look for labs that have all the instruments for us. Once I’m sure I’ve identified Phytophthora, I can use the cultures for my downstream experiments.”

Adarsha and Dr. Whitehill standing next to azure c300
Adarsha and Dr. Whitehill with Azure 400 imaging system in their lab.

Several scientists at North Carolina State University are not letting this pathogen get in the way of Christmas tree production. For PhD student Adarsha Devihalli, the solution is in the molecular details. His research focuses on studying a particular strain of Phytophthora and its genetic code. His initial work focused on pathogen identification. In this next phase of his research, he will use functional genomics tools to enable the identification of genes in the pathogen important for the initiation of the infection process.

Devihalli isn’t the only one working on Phytophthora, either. He is a member of the Christmas Tree Genetics (CTG) Program, headed by Dr. Justin G. A. Whitehill, Assistant Professor and Director of the Christmas Tree Genetics Program at NC State University.

Under the guidance of Dr. Whitehill, Devihalli is studying this devastating disease to better understand the issues at hand. Together, Whitehill CTG lab members are working towards the development of novel genomic resources for Fraser fir to combat several pests of these celebrated trees.

How the samples are collected

To begin his experimental process, Devihalli first visits the NC Department of Agriculture’s research station in Ashe County – located approximately four hours away from the university in Raleigh. He looks for disease-related symptoms on Fraser firs, collects samples, and returns to the lab for culturing, identification, and analysis using the Azure 400 Imaging System.

Azure 400 Visible fluorescent imaging system
The Azure 400 is capable of three-channel visible fluorescence detection, which enables sensitive multiplex detection of Western blots, fluorescent biomolecules and Cy2/Cy3/Cy5 or similar fluorochromes. This fluorescent imager allows users to simultaneously image and quantify up to three different targets.

Looking to the future

Together, the Whitehill CTG lab and Devihalli intend to use their experimental results to help further current knowledge of the Fraser fir genome, and uncover potential genetic resistance mechanisms to Phytophthora root rot.  Ultimately, they plan to develop better mitigation methods for root rot in the country’s most beloved Christmas tree.

“At present, there is no publicly available sequencing information for these species,” explains Devihalli. “We don’t have a genome sequence for Fraser fir, so this is a big goal for our lab [yet].”

DISCOVER: Azure 400 Imager

For more information on Dr. Whitehill’s Christmas tree research at NC State, visit https://research.cnr.ncsu.edu/sites/whitehilllab/

Quantitative Western Blot Quiz

Categories
Western Blotting

True or False: To get quantitative Western blotting data do all of the following:

• Follow your typical Western blotting protocol
• Be sure to probe for your protein-of-interest and a housekeeping protein so you can normalize your data
• Image the blot on a digital imager
• Draw boxes around the bands of your protein-of-interest and your housekeeping protein and use the imager to generate a number for band intensity
• Follow your imager’s instructions for subtracting background
• Calculate the ratio of your protein-of-interest to housekeeping protein to obtain relative protein abundance






What’s your quantitative western blotting IQ?

[qsm quiz=1]

Are you doing everything you should to ensure accurate quantitative Western blot data? Find out by testing yourself with this quantitative western blotting quiz. If you get one or more questions wrong, you can brush up on the basics by downloading our Quantitative Western Blotting Basics guidebook using the form on the right.

1. True or False: To get quantitative western blotting data do the following:
  • Follow your typical western blotting protocol. Be sure to probe for your protein-of-interest and a housekeeping protein so you can normalize your data
  • Image the blot on a digital imager
  • Draw boxes around the bands of your protein-of-interest and your housekeeping protein and use the imager to generate a number for band intensity. Follow your imager’s instructions for subtracting background
  • Calculate the ratio of your protein-of-interest to housekeeping protein to obtain relative protein abundance
 
2. True or False: You must use fluorescently-labeled antibodies to get quantitative western blotting data.
3. Which of the following methods can you use to validate an antibody for quantitative western blotting:

A. Genetic method: Show that when the amount of your protein-of-interest is reduced, the signal from your antibody used in an ELISA assay is also reduced.

B. Orthogonal method: Show that measurement of protein abundance using your antibody correlates strongly with the measurement of protein abundance using an orthogonal method such as mass spectrometry.

C. Independent antibody: Show that the measurement of protein abundance using your antibody correlates strongly with the measurement of protein abundance using a second, already validated antibody.

4. True or False: The best way to normalize western blot data is to use a housekeeping protein?
5. Which of the following parts of the western blotting workflow should be tested to ensure that your experimental conditions are not causing the signal to saturate:
SOURCE
  1. Janes KA. An analysis of critical factors for quantitative immunoblotting. Sci Signaling. 2015 Apr 7;8(371):rs2. PMCID: PMC4401487.
  2. Uhlen M, et al. A proposal for validation of antibodies. Nat Methods. 2016 Oct;13(10):823-7. PMID: 27595404.
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.

Investigating the Mechanism of Pathology in Parkinson’s Disease

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Imaging Publication Spotlight

Parkinson’s disease (PD) is an age-related neurodegenerative disorder that affects almost 1 million people in the United States. PD is associated with motor symptoms including tremors and stiffness that affect balance and coordination. Symptoms appear as dopaminergic neurons are lost from the midbrain. Neuronal death appears to be due to the accumulation of toxic aggregates, called Lewy bodies, of the protein alpha-synuclein in neurons.

In recent work, Anandhan et al investigated the role of a transcription factor, NRF2, in alpha-synuclein driven pathology. NRF2 is known to regulate the cellular stress response and lack of NRF2 has been shown to exacerbate PD pathology, but the precise roll of NRF2 in alpha-synuclein driven pathology is unknown. The authors created mice that overexpress human alpha-synuclein as a model of PD. They then knocked out the gene coding for NRF2 so study the effect of the loss of NRF2 on behavioral tests, neuron loss in several brain regions, and phosphorylation and oligomerization of alpha-synuclein.

The research compared 4 groups of mice:

  1. Expressing human alpha-synuclein, expressing Nrf2 (ha-Syn+/Nrf2+)
  2. Expressing human alpha-synuclein, Nrf2 knockout (ha-Syn+/Nrf2-)
  3. Not expressing human alpha-synuclein, expressing Nrf2 (ha-Syn-/Nrf2+)
  4. Not expressing human alpha-synuclein, Nrf2 knockout (ha-Syn-/Nrf2-)
immunoblot
Figure 1. Generation of a novel humanized α-Syn/NRF2 mouse model of PD. (A) Mice overexpressing human wild-type α-Syn (hα-Syn+) were initially cross bred with Nrf2 knockout (Nrf2-/-) mice to result in hα-Syn+/Nrf2+/- and hα-Syn-/Nrf2+/- mouse strains. The hα-Syn+/Nrf2+/- mice were further crossed to finally generate four genotypes: hα-Syn+/Nrf2+/+, hα-Syn+/Nrf2-/-, hα-Syn-/Nrf2+/+ and hα-Syn-/Nrf2-/- strains. Mouse genotypes were confirmed by PCR (B and C) of tail DNA and Western blotting using Azure c600 (D).

According to the study, lysates were boiled, sonicated, and resolved by SDS-PAGE. Membranes were subjected to appropriate antibodies at 4°C for overnight before being incubated with anti-mouse or anti-rabbit horseradish peroxidase (HRP) conjugated secondary antibodies from Sigma Aldrich and imaged using the Azure Biosystems c600 imager (Figure 1D).

Since the release of this publication, the Azure c600 has been upgraded to the Azure 600. The Azure 600 is the only system that offers two channel, laser-based IR and chemiluminescent detection, with the speed and sensitivity of film, with the ability to image visible fluorescent dyes, standard EtBr and protein gels, and infrared laser excitation for quantitative Western blot imaging in the NIR.

The Ultimate Western Blot Imaging System

The Azure 600 offers laser technology with two IR detection channels enabling you to image more than one protein in an assay. It provides accurate and fast chemiluminescent detection, as well as the sensitivity, dynamic range, and linearity needed for quantitative blot analysis.
Scientist choosing settings on Azure 600
The Azure 600 is the only system that offers two channel laser based IR detection, chemiluminescent detection with the speed and sensitivity of film, and the ability to image visible fluorescent dyes, standard EtBr and protein gels.

Findings suggest activating NRF2 might present a way to delay PD progression

The study found increased alpha-synuclein phosphorylation and oligomerization in the mice expressing human alpha-synuclein and lacking Nrf2. These mice also lost tyrosine hydroxylase–expressing dopaminergic neurons in the substantia nigra and had behavioral defects consistent with early-state PD. The authors conclude that loss of NRF2 drives development of alpha-synuclein related PD pathogenesis via effects on oxidative stress, proteostasis, inflammation, and cell death. They suggest activating NRF2 might present a way to delay onset or progression of PD.

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How the Sapphire Scanner is Used to Better Grocery Store Tomatoes

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Imaging Publication Spotlight Western Blotting

Few things are more disappointing than a tasteless, mealy grocery store tomato. These bland fruits are pale imitations of the vine-ripened tomatoes available from the garden at the end of summer. Tomatoes are perishable, and providing ripe, high-quality tomatoes that maintain their texture and nutritional content is a challenge for commercial growers.

recent publication by Tsafouros et al from the Institute of Olive Tree in Greece provides a window into the intense, ongoing research aimed at understanding how and why tomatoes and other fruits ripen under various conditions. Such studies could improve the postharvest shelf-life and the quality of commercially grown tomatoes.

Growing the tomatoes used in this study

For this study, tomatoes were grown in a greenhouse and either picked at “commercial maturity” (when the tomato is just turning color) or left on the plant to mature for an additional week. The harvested tomatoes were stored at 5, 10, or 25 °C and after 7 days were compared to each other and to tomatoes left to ripen on the vine.

Tomatoes are typically picked before ripening and stored and transported at low temperatures in an attempt to increase the shelf-life. Tsafouros et al examined the effect on ripening of storing picked tomatoes at various temperatures for a week. The authors also characterized in detail the effect of storage temperature on the metabolism of polyamines, compounds known to play a role in fruit ripening and the content of which are known to be associated with tomato quality and shelf life.

The role of temperature in mediating postharvest polyamine homeostasis in tomato fruit
Chemiluminescent Western blot showing the role of temperature in mediating postharvest polyamine homeostasis in tomato fruit

How the research was conducted

The authors carried out an exhaustive biochemical and molecular biological characterization of polyamine metabolism in the tomatoes. They assessed the total content of a variety of polyamines, the activity of the enzyme responsible for breaking down polyamines, the expression of all 23 genes encoding factors known to be involved in polyamine metabolism, the levels of the proteins involved in polyamine synthesis, and the levels of hydrogen peroxide, a biproduct of amine oxidases acting on polyamines. Protein levels were measured by chemiluminescent Western blots imaged using the 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. Learn more.

Sapphire FL biomolecular imager
The new Sapphire FL images phosphor screens with high sensitivity. With customizable and user-changeable laser and filter modules, it easily adapts to a lab’s changing needs and advancing research.

Learn more about the Sapphire imager and how it can support your research by requesting a quote.

The findings

Their results demonstrate that cold storage alters polyamine metabolism, and support storage of tomatoes at 10°C after picking at commercial maturity. Lower temperatures appeared to induce a stress response, perhaps to protect against chilling injuries, while higher temperatures were associated with lower polyamine levels and lower quality fruit.

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Regulation of Gene Expression by Enhancer RNAs

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Imaging Publication Spotlight 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.

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 is a transcription factor involved in many processes, including cell cycle inhibition and tumorigenesis, and expressed in specific cell lineage. It also plays a role in maintaining cell identity. To study CEBPA protein levels, the authors carried out a quantitative near-infrared fluorescent Western blot imaged on an Azure Sapphire™ Biomolecular Imager (Figure 7). 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.

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 findings

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. 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.

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Requirements of transcription initiation

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 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. Download a free copy of the Sapphire Applications Booklet and learn about how you can add more applications to your arsenal here.

More research done with the Azure Sapphire: