Accurate results for quantitative Western blotting

The ultimate goal of quantitative Western blotting is to determine the precise amount of a protein of interest present within a sample. To accomplish this, the protein of interest is first isolated and then conclusively visualized. Researchers typically visualize individual proteins after affixing a signal-emitting marker—usually a tag on an antibody—to them. The emitted signal’s magnitude correlates to the amount of bound antibody, which in turn represents protein abundance.

Early protocols for Western blotting relied on radioligand binding for signal generation, but this was cumbersome, relatively imprecise, and posed safety risks. Chemiluminesence proved a very popular alternative, and this popularity persists to this day. However, recent advances in optical imaging technology and the development of more sensitive labels^1,2 have facilitated the adaptation of fluorescence imaging techniques for Western blot visualization and analysis.

Chemiluminescence is broadly defined as a chemical reaction resulting in the emission of light. In Western blotting protocols, this reaction is engineered by introducing both an enzyme—usually horseradish peroxidase (HRP)—and the substrate to the protein-bound membrane. Generally, the enzyme is conjugated to a secondary antibody designed to bind specifically to protein-bound primary antibody. This ensures that the amount of HRP—and therefore the amount of light generated—correlates only with the amount of protein of interest and no other non-specific elements.

HRP catalyzes the reaction between luminol and an oxidant (e.g., hydrogen peroxide), forming an electronically excited 3-aminophthalate intermediate. The decay of this intermediate to stable 3-aminophthalate emits low-level fluorescence at 450 nm.^3,4 Commercially available enhanced chemiluminescence (ECL) kits provide solutions containing not only substrate and oxidant, but also an enhancer molecule (e.g., phenol, naphthol, aromatic amine, or benzothiazole)⁴, resulting in increased light emission and significantly delayed signal decay.³ Emitted light is then visualized either using X-ray film or a camera.

Chemiluminescence- and ECL-based methods are straightforward, necessitating only an HRP-conjugated antibody and a luminol reagent solution–both of which are readily available from commercial manufacturers. Visualization and imaging, whether using film (which is then digitally scanned) or camera-captured digital images, have relatively low technical requirements. This simplicity is likely a contributing factor to the popularity of the modality.

However, chemiluminescence does possess several limitations. First and foremost, it provides an indirect assessment of protein quantities. Detected light signal does not linearly correlate with protein abundance at amounts greater than 5 µg.² This is likely due to the potential for multiple secondary antibodies to bind to a single primary antibody. Additionally, chemiluminescence-based techniques have a relatively narrow dynamic range and have been called “semi-quantitative”.⁵ Signal saturation is a particular problem, especially for ubiquitously expressed housekeeping proteins, as each HRP bound to a secondary antibody has multiple binding sites for interaction with ECL substrates. This exponential signal amplification leads to a rapid plateau.

Fluorescence-based techniques for Western blotting were developed in response to these issues. Here, similar to methods used for flow cytometry and immunofluorochemistry, signal emitted by fluorophore-tagged secondary antibodies is detected and measured.² Fluorophore signal more linearly correlates with protein concentration,⁵ likely because it is a more direct assessment of protein abundance than enzymatic function. It also provides a greater dynamic range, offering superior sensitivity for both low and high protein amounts.^1,5

Additionally, fluorescence-based methods facilitate multiplexing, allowing researchers to probe two or more proteins simultaneously. This means that researchers can avoid time-consuming stripping techniques which can potentially remove protein from the membrane, as well as concerns about signal overlap or over-/undersaturation. The latter is particularly useful when quantifying housekeeping proteins, which are likely many times more abundant than experimental proteins of interest.

However, fluorescence-based Western blotting protocols can present logistical issues. Researchers seeking the most out of fluorescence need to account for both source species and fluorophore excitation/emission wavelength when selecting a secondary antibody. In contrast, chemiluminescent methods typically need only a single HRP-conjugated secondary antibody targeting the source species of the primary antibody. Additionally, in contrast to film-based light capture, detecting fluorescence signal requires a specialized digital imager, ideally with multiple filters to maximize multiplexing potential.

Chemiluminescence-based Western blot protocols have been a laboratory staple for many years, but methods utilizing fluorescence have become more prevalent as researchers seek increased quantitation accuracy. Fluorescence-based strategies do have greater infrastructural requirements, but offer superior signal sensitivity, a broader dynamic linear range, and multiplexing potential. Exploring fluorescence may be a worthwhile pursuit for researchers needing more precision from their Western blots.

References

1. S.L. Eaton et al. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting. PLoS One. 2013 Aug 30;8(8):e72457.

2 S.L. Eaton et al. A guide to modern quantitative fluorescent western blotting with troubleshooting strategies. J Vis Exp. 2014 Nov 20;(93):e52099.

3 R.A.W. Stott. "Enhanced Chemiluminescence Immunoassay." The Protein Protocols Handbook (2009): 1835-1843.

4. G.H. Thorpe and L.J. Kricka. Enhanced chemiluminescent reactions catalyzed by horseradish peroxidase. Methods Enzymol. 1986;133:331-53.

5. M. Zellner et al. Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples. Electrophoresis. 2008 Sep;29(17):3621-7.