Western blotting is a technique routinely used by researchers to detect and identify certain proteins of interest in a sample. It is typically performed in complex samples, such as lysates from cells or tissues, to monitor the protein expression as a response to treatment, or in plasma samples, to validate biomarkers. Moreover, Western blotting is a useful bioanalytic tool in purification procedures, as it controls the protein presence and stability during the process. Although a well-established and explored method, it is still challenging to obtain consistent and quantifiable results using Western blotting. A number of shortcomings need to be addressed to enable Western blotting to deliver standardized and quantitative data, supporting researchers who require reproducible, high-quality results to move their research forward.
Western blotting workflow includes many hands-on steps, reagents and choices of imaging methods, which in turn contribute to the challenges of obtaining precise data. The procedure involves applying samples to gel electrophoresis for protein separation and immobilization of the proteins to a membrane following electrophoretic transfer from the gel. Non-protein binding areas on the membrane are “blocked” with a generic protein, often milk or bovine serum albumin, to prevent non-specific binding of antibodies. The membrane is then incubated with a primary antibody that specifically binds to the target protein of interest. Unbound antibodies are removed by washing, and a labelled secondary antibody conjugated to an enzyme, fluorophore or an isotope is used for detection. The signal emitted by the protein and antibody complex is proportional to the amount of protein, thus enabling quantitation.
The majority of researchers currently use chemiluminescent detection methods. This approach is supported by a wide range of suitable reagents and supports versatile imaging options, as CCD cameras or X-ray film can be used to capture the light output. Because this relies on the indirect signal obtained from the reaction between the enzyme and substrate, the light signal produced is unstable and declines over time. This results in signal intensity variation between blots, giving inaccurate and poorly reproducible densiometric data.
Similarly, strong light signals can cause X-ray film to become saturated and the quantity of protein cannot be accurately determined, as there is no longer a linear relationship between the signal and amount of protein. In addition, chemiluminescence can only be used to detect one protein at a time. Given there are a number of proteins that may need to be detected from one sample, stripping of the antibody and re-probing is required to detect a second protein, which all add extra steps and additional variation in results.
To help address these challenges, researchers are increasingly looking to use fluorescent Western blotting whereby a fluorophore on the secondary antibody directly produces an output— no longer relying on the reaction of chemical reagents as in chemiluminescence. Fluorescence signals are highly stable and can be multiplexed using different fluorophores for different antibodies, allowing multiple proteins to be detected simultaneously. The consistency of the signal intensities allows repeat exposures and low detection limits across a broad dynamic range, quantifying both high- and low-expressed protein levels. Therefore, fluorescent Western blotting simplifies the workflow and is a more efficient alternative to more traditional methods.
One final challenge to address is the complications that can arise during normalization of a target protein signal to the amount of protein applied to the gel, which ensures accurate quantitation by correcting for uneven sample loading. This is commonly done by normalizing the target protein to a stable expressed housekeeping protein— often actin, tubulin and GAPDH are used, as they are highly abundant in cells. In some cases, when a large quantity of sample is required to detect a less abundant target protein, the housekeeping protein will give a very intense signal, which skews the linear relationship between signal and amount of protein, giving inaccurate quantitation and inconsistent results.
To avoid this scenario, researchers can normalize against the total protein content in the sample instead. However, at optimized conditions, housekeeping proteins provide a linear response and it is then a viable option to utilize the multiplexing capability offered by fluorescence detection, housekeeping protein and target proteins can be simultaneously detected.
In conclusion, it is key that the challenges in obtaining signal linearity and a dynamic range of detection and normalization are addressed to enable Western blotting to realize its potential as a method for accurate, reproducible and reliable quantitation of proteins. It is likely this will be achieved by a combination of a greater understanding of the limitations of current methods, coupled with advances in the technologies being used to standardize the approaches used.