Western blot is viewed by many as the simplest and most convenient technique for identifying individual proteins within a complex mixture. It has come a long way in the past ~40 years, with the early use of radioisotope-labeled secondary antibodies having been superseded by enzymatic and fluorescent detection. In this article, we explain why western blot retains its appeal and provide some tips for selecting a suitable detection method.
Western blot offers many advantages
According to a review published in early 2020, western blot has been mentioned in the titles, abstracts and keywords of more than 400,000 PubMed-listed publications. “This reinforces its reputation as a well-established and accessible technique,” says Hanna Dreja, team leader of the western blot core facility within new product development at Abcam. “It also highlights the sustained attraction of a method that does not necessarily require expensive reagents and equipment to generate reliable results.”
Tobias Polifke, co-founder and managing director at CANDOR Bioscience, agrees, adding that a main advantage of western blot is that it can be used to evaluate almost any type of sample matrix or analyte, including fat tissue and membrane proteins that are often challenging to analyze with other methods. “Western blot is both flexible and robust in its application,” he explains, “and, in my opinion, its use is unlikely to reduce in volume in the near future.”
Different detection methods for different needs
Western blot detection can broadly be categorized as enzymatic—using either a chromogenic or chemiluminescent substrate—or fluorescent. “Chromogenic western blot detection is rarely used these days due to its low sensitivity and the irreversible nature of the precipitation reaction,” notes Jeff Harford, senior product marketing manager at LI-COR Biosciences. “A further drawback of this method is that it is highly subjective, since the end user defines the length of time taken for color to develop. These factors tend to limit chromogenic western blot detection to the qualitative assessment of abundant protein targets, with chemiluminescence and fluorescence being preferred alternatives.”
Chemiluminescent western blot detection is best known for its exquisite sensitivity—a feature that makes it the most common form of detection. Provided an appropriate substrate is used, low femtogram amounts of protein can be identified by chemiluminescent western blotting. Unlike chromogenic methods, chemiluminescent detection allows the membrane to be stripped and re-probed. However, Dreja cautions that this can reduce the signal and stresses the importance of including relevant controls to confirm that proteins have not been lost during the process. It is also critical that the blot is imaged immediately after addition of the chemiluminescent substrate to capture the enzymatic reaction as it takes place.
Fluorescent western blot detection benefits from a broad dynamic range and the capacity for multiplexing. “The fluorescent signal is also very stable, meaning that blots can be reimaged after several months when they are stored correctly,” reports Min Teng, quality inspector at Sino Biological. According to Harford, the sensitivity of fluorescent detection is comparable to that of chemiluminescence where imaging is performed in the near-infrared spectrum. He also comments that fluorescent western blot detection provides more accurate quantitation than chemiluminescent detection because it is independent of an enzymatic reaction that can vary with time, temperature, and substrate consumption.
“A further important benefit of fluorescent western blot detection is that it affords significant savings in terms of both reagents and time compared to chemiluminescent detection,” explains Martin Miguez, product development manager at Azure Biosystems. “A typical two-target enhanced chemiluminescent (ECL) western blot for a protein target and its post-translational modification could cost around US$ 40 more and take twice as long as fluorescent western blotting due to the need for stripping and re-probing. Moreover, with the availability of instrumentation to image fluorescent western blots in three or four independent channels, researchers can now detect and quantify three different targets plus the total protein population of a sample in a single experiment, which is a considerable improvement on early forms of the western blotting technique.”
New technologies enhance Western blotting
The practice of using total protein staining for normalization is one of many innovations designed to increase the value of western blot data. Polifke remarks that reagents for one-step immunodetection dramatically reduce the background seen with blocking reagents such as milk or BSA, while Dreja suggests that the need to multiplex western blotting has introduced some additional players to the field.
“Platforms for automated western blotting are now widely available, including Simple Western™ and DigiWest®,” she says. “These technologies are attractive modern alternatives to the classic method, not least because of their small sample volume requirement and higher throughput. But researchers should always bear in mind that for any form of western blot to be successful, the specificity and selectivity of the antibody reagents are pivotal.” Lastly, Harford adds that some suppliers have made improvements to the software used for analyzing western blots that have also contributed to better data quality. “Next-generation software such as Empiria Studio is aligned with best practices put forward by publishers and granting agencies,” he says. “In turn, this has greatly reduced inter-operator variability, leading to more accurate assessment of protein expression by this tried and trusted technique.”
Tips for selecting the right Western blot detection method
The following tips combine the feedback provided by all five contributors to this article:
- Find out what equipment you have available—if you have access to an imager, establish what forms of detection it supports
- Understand your target—if your protein is expressed at low levels, chemiluminescent detection with a high sensitivity substrate can be a sensible option; if you wish to analyze a protein and its phosphorylated isoform simultaneously, fluorescent detection is a better choice
- Ask other researchers what detection method they are using for your protein of interest
- Consider using total protein staining rather than a housekeeping protein to normalize data; determine whether your imager can measure this
- Think about using reagents that enable one-step Western blot detection, such as ReadyTector® technology
- For quantitative analysis of western blot data, consider using fluorescent detection rather than enzymatic detection where the reaction can be influenced by factors such as temperature and time
- If you wish to keep your membrane for future scanning, choose fluorescent detection
- Refer to publisher best practices for generating and analyzing western blot data and incorporate these into your workflow where possible
- Choose a detection system that offers flexibility, such as an imager that can be upgraded with additional detection modalities to suit future needs
- If you plan to invest in new western blot detection reagents or equipment, ask suppliers to explain the features and benefits of a particular product; when purchasing an imager, consider not only the price of the instrument but also the western blotting throughput and the cost of the reagents—savings made here might cover the price of a high-end instrument in a short period of time