Western Blot Image Analysis

When performing western blot image analysis, researchers may be tempted to follow the same protocols that have always been used in their lab. But with publication guidelines becoming stricter, it’s worth keeping abreast of developments in the field, many of which can improve data accuracy.

Basic principles of western blot image analysis

Western blotting begins with separating a mixture of proteins on a gel, transferring them to a membrane, and detecting one or more targets of interest with labeled antibodies. An image of the membrane is then captured—either on to film or using a digital imager—to allow for analysis. Typically, the analysis process is quantitative, whereby the signal is measured, the background subtracted, and normalization performed to confirm that any change is due to the biology and not experimental error. Yet while this all sounds fairly straightforward, there are numerous places where mistakes can be made.

“Critically, researchers should never simply assume that the relationship between signal and protein amount is linear,” says Matthew Hammond, Protein Quantitation Applications Manager at Bio-Rad. “Many steps of the western blotting process can be non-linear, meaning that best practices are all aimed at ensuring that you are working within the linear dynamic range of the assay from start to finish.”

Best practices ensure accurate results

So, what are some of the best practices you should employ to accurately quantify your western blot data? Jade Fee, Application Scientist at Azure Biosystems, notes that optimizing primary and secondary antibody concentrations and incubation times is key. “As well as minimizing non-specific binding that can cause unwanted background, antibody optimization can help prevent the signal from becoming saturated,” she says. “Further ways of avoiding image saturation include not overloading the gel and using an appropriate exposure time. With digital imaging, it is easy to detect pixel saturation, as well as to capture multiple images of a blot to find the exposure time that minimizes background while maximizing specific signal.”

Other recommendations for ensuring data accuracy include using proper controls to confirm antibody specificity and validate results, and keeping your experiment consistent. “The latter is probably the most difficult, but the most important, factor in ensuring reliable results,” reports Afrida Rahman-Enyart, Ph.D., Scientific Liaison and Product Manager at Proteintech. “You want to make sure that between replicate experiments, factors including tissue culture, lysate preparation and storage, gel selection, voltages, buffers, washes, antibody dilutions, and so forth are all as consistent as possible. This will minimize variations between replicates and allow you to get a more accurate representation of your data.”

“Once you have captured your image, working through the data analysis steps methodically is essential,” adds Hammond. “These steps broadly comprise identifying which pixels in your image belong to your bands, figuring out how much of the measured light in those pixels comes from your protein signal, and comparing your target signal to a loading control signal in the same lane. While such actions can be almost trivial if you have nice, bright bands, more thoughtful application is required if you are working with weak signals or uneven backgrounds. For example, leaving in too much background or excluding too many of the pixels that belong to your band of interest can skew your results.”

Modern advances improve data accuracy

Within the past few decades, western blot image analysis has been improved by various advances. For researchers still using film, freeware such as ImageJ from the National Institutes of Health (NIH) has enabled quantification of protein bands relative to a conventional loading control (i.e., a housekeeping protein) without the need for financial outlay, while for those who have switched to using a digital imager, numerous benefits are on offer.

“Digital imaging has improved the image analysis process in several ways,” comments Hammond. “As well as enabling researchers to quickly and easily optimize exposure times to avoid image saturation, digital imagers provide the flexibility to prioritize sensitivity, speed, or resolution during image acquisition, making the overall process more user friendly for different applications.” Other advantages of digital imaging systems include the ability to automate fundamental processes such as band detection and background subtraction, which can reduce inter-operator variability, and the capacity to store images and experimental metadata away from the instrument, which can streamline workflows and promote collaboration.

The advent of fluorescent detection has also brought many benefits to western blotting. “One of the main advantages of fluorescent detection is that it allows for multiple proteins to be detected on the same membrane without the need for stripping and re-probing,” says Rahman-Enyart. “This is valuable when looking at protein interactions or changes in multiple protein levels between conditions, since stripping and re-probing not only costs time but can also introduce errors if it is not performed correctly.” To simplify multiplexing, researchers may choose to use fluorophore-labeled primary antibodies, which they can generate in-house with products such as Proteintech’s FlexAble Kits if an off-the-shelf conjugate is not readily available.

Western blot detection with labeled primary antibodies. siRNA transfected HEK293 cell lysates were detected with anti-GNA13 (67188-1-Ig) labeled with FlexAble CoraLite^® Plus 555 Kit (KFA022,green) and anti-PCNA (60097-1-Ig) labeled with FlexAble CoraLite^® Plus 647 Kit (KFA023, red). Image provided by Proteintech.

Another advantage of fluorescent detection is that it can be more quantitative compared to methods based on chemiluminescence, as the fluorescent signal is linearly proportional to the amount of antibody bound to the blot. “Because chemiluminescence depends on an enzymatic reaction, it can become non-linear when the substrate is depleted,” explains Fee. She also comments that near infrared (NIR) detection, which is possible with the Azure Imagers and the Sapphire FL Biomolecular Imager, introduces the additional benefits of lower non-specific background and the ability to detect as many as four proteins on one blot by expanding the fluorescent probe compatibility into the visible fluorescent range.

Total protein staining is another important western blotting innovation that is now considered the preferred loading control. “While it was once common for researchers to normalize western blot data to a housekeeping protein, this is now seen as non-ideal for several reasons,” says Fee. “First, the housekeeping protein and the protein of interest are often not expressed at similar levels, making it difficult or impossible to find an amount of sample to load on the gel in which both proteins can be detected within the linear range of the experiment. Second, evidence now suggests that many housekeeping proteins are not expressed at constant levels across tissue types and may be affected by the experimental manipulation being studied. Consequently, total protein staining—a method that uses the entire population of proteins in each sample for normalization—is considered a more reliable loading control, as this measurement is subject to less experimental variability.”

Total protein staining with TotalStain Q. Each well contains 5 μg HeLa lysate. TotalStain Q imaged in Cy3 channel (green), hnRNP K and molecular weight marker imaged in 700 nm channel (red), GAPDH imaged in 800 nm channel (blue). Image provided by Azure Biosystems.

Future perspectives

With western blotting maintaining its status as one of the most popular immunoassay techniques, researchers and manufacturers are united in their efforts to increase its utility. Rahman-Enyart notes that knockout/knockdown models are seeing increased use for antibody validation, which can lead to cleaner blots, while Fee suggests that being able to image the same blot in multiple modes on a single instrument expands analysis possibilities. Finally, Hammond comments on the value of educational outreach programs, like the Biocompare editorials. “Just because western blot is simple does not mean it is easy,” he says. “There is a lot of subtlety in the art of collecting western blot signals, and helping users identify the pitfalls and best practices will increase the quality of western blotting data throughout the field.”

Key Takeaways

• Never assume that the relationship between signal and protein amount is linear.
• Remember to optimize antibody concentrations and incubation times to minimize background and prevent signal saturation.
• Always include proper controls to confirm antibody specificity and validate results.
• Be consistent between replicate experiments to safeguard reproducibility.
• Work through the analysis steps methodically to ensure data are correctly interpreted.