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.
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.
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. Let’s review the cases where you would use either application.
When to use ELISA for protein analysis
| 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. |
Table 1. Advantages of measuring levels of specific proteins by ELISA
| Advantages | Disadvantages |
|---|---|
| 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. |
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 new Sapphire FL, you are able to save precious time (and $$$) on each multiplex Western blot. Don’t just take our word for it, compare the costs here.
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.
| 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. Advantages of using Western blotting over ELISA
Above are 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.
| Advantages | Disadvantages |
|---|---|
| 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 Reader. The amount of signal produced is directly proportional to the amount of target protein in the sample. The Ao conveniently comes with 8 filters: 5 standard filters (405, 450, 492, 595, and 630 nm), and 3 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 (shown below), an HRP-conjugated secondary antibody binds to the primary antibody. When exposed to a detection substrate like ECL, light is produced. This can be detected two ways: 1) using a chemiluminescent imager like the new chemiSOLO, or 2) in the darkroom using film. Fluorescent Western blotting uses secondary antibodies directly conjugated to fluorescent dyes. Fluorescent Western blots are typically only visualized using digital imagers, such as the Azure 400.
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.
Revolutionizing the Way You Western blot
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. Narrow, specific laser excitation leads to higher signal, with less background and increases the overall sensitivity of your Western blots.
Choose 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, an 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.
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