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. 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.

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.

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 Reader from Azure Biosystems. The Ao comes with 8 filters: 5 standard filters (405, 450, 492, 595, and 630 nm), and 3 filters you can personalize. The ability to choose your own filters ensures you have what your assays require. The amount of signal produced is directly proportional to the amount of target protein in the sample.

Azure Ao Microplate 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.

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 fluorescence.

Chemiluminescent Western Blot imaged with Azure Imager
Chemiluminescent Western blot imaged with Azure Imager

For chemiluminescence, an HRP-conjugated secondary antibody will bind the primary antibody. When exposed to a detection substrate, like ECL, light is produced and can be detected using a chemiluminescent imager like the new chemiSOLO, or in the darkroom using film. Fluorescent Western blots are typically visualized using an Azure 400, or other digital imager. The newer generation of imaging systems often contain sophisticated cameras that exhibit a broader dynamic range than film, thus avoiding the signal saturation problems that limit the dynamic range of film.

Whatever your Western blot needs are, we can help! If you’re unsure of which system you need, an Azure Biosystems expert is available to help you choose the right option. Fill out the form on this page and let us know what you’re looking for in a system. That’s it. We’ll match you up with the best fit for your research.

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 ELISA and Western blotting are important techniques for detecting and quantifying proteins. Each technique has its own unique advantages and disadvantages, making each suitable for different applications. Let’s review the advantages and disadvantages of both applications.

Depiction of chemiluminescent Western blot signal
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.

Advantages and Disadvantages of using ELISA for protein analysis

High sensitivity: One of the major advantages of ELISA is its high sensitivity, making it capable of detecting very small amounts of target proteins, even in complex mixtures. This makes it an ideal choice for detecting rare or low-abundance proteins.Limited multiplexing capability: Traditionally, only one protein can be targeted in each ELISA. This makes it difficult to simultaneously analyze multiple proteins in a single sample. (Read more about multiplex western blots)ce
Quantitative analysis: ELISA can be used to measure the exact amount of target protein in a sample, making it a valuable tool for determining protein concentration or monitoring changes in protein levels over time.High background: ELISAs can have high background due to cross reactivity of primary and secondary antibodies.
Rapid and easy to perform: ELISA can be performed relatively quickly and requires minimal sample preparation, making it a convenient choice for routine assays.
Versatile: ELISA can be used for both qualitative and quantitative protein analysis on a wide variety of sample types, including serum, cell culture supernatants, and urine. Since they are usually performed in 96-well plates, ELISAs allow for many conditions and repeat samples to be tested at once.
High-throughput: Due to being performed in a 96-well plate, more samples can be processed faster and with less sample needed. Check out this report of an ELISA being performed using a 384-well plate from the National Institute of Health.


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!

Advantages and Disadvantages of Western blotting for protein analysis

ELISA is a high-throughput, sensitive, and simple assay that is ideal for detecting low-abundance proteins in large numbers of samples. On the other hand, 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.

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.

When to use ELISA vs Western blot

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, an Ao Microplate Reader or Azure 400 Imaging System. If you’re unsure about what you need for protein analysis, fill out this form and a product expert will be in touch!

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