When to use ELISA vs Western blot

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ELISA (Enzyme-linked immunosorbent assay) and Western blotting are two of the most commonly used techniques in molecular biology for the detection and quantification of proteins. Although both techniques are based on the principle of binding specific antibodies to target proteins, they have different applications and advantages.

By weighing the pros and cons of both ELISA and Western blotting for protein analysis, you can make a more informed decision about which method is best for your experiment. Both are important techniques for detecting and quantifying proteins, but each technique has its own unique advantages and disadvantages.

In this blog post, we will discuss the key differences between ELISA vs Western blotting to help you decide when it is best to use each method.

Table of contents

What's the difference between ELISA and Western blot?

ELISAs are immunoassays carried out in 96-well plates as a simple, rapid, and highly sensitive method for measuring the presence of a specific protein in a sample. ELISAs are often used for qualitative and quantitative analysis of proteins in various sample types, such as serum, cell culture supernatants, and urine.

Western blotting is a powerful technique for the detection and characterization of specific proteins in a sample. The basic principle of Western blotting involves separating proteins in a sample using electrophoresis, transferring them to a nitrocellulose or PVDF membrane, and then probing the membrane with an antibody specific to the protein of interest. If the target protein is present, the antibody will bind to the protein. A secondary antibody allows for detection through one of two main methods: chemiluminescence or fluorescenceLet’s review the cases where you would use either application.

When to use ELISA for protein analysis

Now that we have covered the instances where you might find using ELISAs more beneficial over Western blots, let’s briefly discuss the disadvantages using ELISA may pose.

ELISAs have limited multiplexing capability; traditionally, only one protein can be targeted in each ELISA. This caveat makes it difficult for ELISA to simultaneously analyze multiple proteins in a single sample. In this case, we suggest using multiplex fluorescent Western blots. Using a fluorescent scanner like the Sapphire FL, you will save precious time (and $$$) on each multiplex fluorescent Western blot. Don’t just take our word for it, compare the costs here.

Table 1. Advantages of measuring levels of specific proteins by ELISA

Use ELISA when you...
Are looking to detect very small amounts of target proteins, or rare or low-abundance proteins. A major advantage of ELISA is its high sensitivity.
Need to measure the exact amount of target protein in a sample. ELISA is a valuable tool for determining protein concentration or monitoring changes in protein levels over time.​
Are in a time crunch in the lab. ELISAs are rapid, easy to perform, and can be done relatively quickly with minimal sample preparation. It is a convenient choice for routine assays.​

When to use Western blotting for protein analysis

Since ELISA is a high-throughput, sensitive, and simple assay, it’s ideal for detecting low-abundance proteins in large numbers of samples. So, when should you use Western blotting for protein analysis? Western blotting is a highly specific technique that is capable of resolving proteins into different molecular weight ranges, making it useful to detect possible protein modifications and subtle differences between experimental conditions.

Table 2. Advantages of using Western blotting over ELISA

Use Western blotting when you...
Need to detect a wide range of proteins. Western blots are valuable tools for analyzing complex mixtures, such as cell lysates. To take your analysis one step further, fluorescent multiplexing will allow you to detect multiple targets at once. The Azure 400 imager allows you to simultaneously detect 3 target proteins image using three-color fluorescence.​
Want to identify specific proteins. Western blots can be used to specifically identify the presence of a target protein in a sample, even when the protein is present at low levels, or in a complex mixture.​
Want to confirm the correct protein is being measured. Western blots can confirm proteins through a number of factors, such as protein size. It can also reveal if there are protein modifications, impurities, etc.

Table 2 shows the instances where you would use Western blotting over an ELISA test; however, keep in mind that unlike ELISA, Western blotting is a multi-step process that can take from several hours to overnight to perform. Western blotting is also less sensitive than ELISA. If you’re working with low-abundance proteins, stick with ELISA.

Want to run a Western blot but not sure where to start? Check out this checklist for everything you need to run a successful Western blot.

Detect a wide range of proteins: Western blots are valuable tools for analyzing complex mixtures, such as cell lysates. Multiplexing using fluorescence allows for the detection of multiple targets in one experiment. Using the Azure 400 allows you to simultaneously detect 3 target proteins image using three-color fluorescence.Longer process with more steps involved: Western blotting is a multi-step process that can take several hours (often overnight) to perform
Ideal for identifying specific proteins: Western blots can be used to specifically identify the presence of a target protein in a sample, even when it is present at low levels or in a complex mixture.Less sensitive than ELISA: Western blotting is less sensitive than ELISA, making it more difficult to detect low-abundance proteins.
Gives additional information about the protein: A Western blot can confirm the correct protein is being measured by using a number of factors such as protein size. It can also reveal if there are protein modifications, impurities, etc.

How does an ELISA test work?

The basic principle of ELISA involves coating a solid substrate, such as a microplate, with an antibody that specifically binds the target protein. The sample potentially containing the target protein is then added to the coated microplate and allowed to incubate. If the target protein is present, it will bind to the antibody and can be detected using a second antibody conjugated to an enzyme that can cause a color change when a specific substrate is added. This allows for the detection of protein in the sample with a plate reader, like the Ao Absorbance Microplate ReaderThe amount of signal produced is directly proportional to the amount of target protein in the sample.

Azure Biosystems Ao Absorbance Microplate Plate Reader is used for ELISA, Bradford Assays, and more
Trust your data. With the Ao Microplate Reader you will not sacrifice speed for accuracy. With a read speed of <6 seconds, and an accuracy of <0.005 ± 1% (0-3) OD, you will quickly get your results and know they are correct.

The Ao conveniently comes with eight filters: five standard filters (405, 450, 492, 595, and 630 nm), and three filters you can personalize for your needs. We give you the ability to choose your own filters to always ensure you have what your assays require.

Western blot detection methods

Western blots can be visualized using chemiluminescence or fluorescence. In chemiluminescent Western blotting (example shown below), an HRP-conjugated secondary antibody binds to the primary antibody. When exposed to a detection substrate, like ECL, light is produced.

Light can be detected two ways:

  1. Using a chemiluminescent imager (like the chemiSOLO), or
  2. In the darkroom using film.
Chemiluminescent Western Blot imaged with Azure Imager
Chemiluminescent Western blot imaged with Azure Imager

For fluorescent Western blots, you will use secondary antibodies that are directly conjugated to fluorescent dyes. Fluorescent Western blots are typically only visualized using digital imagers, such as the Azure 400.

Multiplex fluorescent Western blot from Azure Biosystems imager
Digital image of 4-color multiplex Western Blot. Using distinct fluorescent and near-infrared targeting antibodies can detect each wavelength and merge them into a four-color multiplex image. No background noise or bleeding between channels.

The newer generation of imaging systems use sophisticated cameras that exhibit a broader dynamic range than film, thus avoiding the signal saturation problems that limit the dynamic range of film. For example, the Azure 600 imager comes standard with a 9.1MP camera which provides high-resolution imaging perfect for publications.

Simultaneously Detect Multiple Proteins

Using lasers for NIR fluorescent imaging sets the Azure Imagers apart from competitors. Our Imagers are the only ones on the market to use lasers.
Two scientists looking at multiplex fluorescent Western blot on Azure 600 Western blot imager

Choosing either method based on the needs of your experiment

When choosing between ELISA and Western blotting, consider the specific needs of your experiment and the questions you are trying to answer. Both techniques are valuable tools in your laboratory arsenal. Selecting the right one will depend on the specific requirements of your experiment. Out of ELISA and Western blot, which method do you most commonly use?

If you find yourself now in need of either a microplate reader or a digital imager, you’re in luck. Azure Biosystems is the leading supplier for life science systems. Check out this page to request pricing on the products mentioned in this post, the Ao Microplate Reader or Azure 400 Imaging System. If you’re unsure about what you need for protein analysis, fill out the form below and a product expert will be in touch. Our experts are available to help you choose the right system. Cheers for now.


New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices? Claim your free Western Blotting eBook!

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