Choosing an Imaging Solution
In any cell-signaling pathway, post-translational modifications and other molecular events occur in a small portion of the involved proteins. Moreover, the event is frequently transient and reversible with participating proteins often present in low numbers. A commonly encountered setback in quantitating the change in expression of the aforementioned proteins, is doing so with an acceptable margin of error. Technological advances have brought about imaging techniques capable of low-signal detection, thereby producing unambiguous protein expression results. Sample preparation, protein mass standardization, gel electrophoresis, and antibody optimization require considerable effort; it is therefore of paramount importance that a visualization method suitable for detecting a low-abundance protein of interest be in place at the finish line.
Western blotting data are based on the immunodetection of target proteins, which must be labeled for visualization by various signal-detection methods. Available imaging solutions are either chemiluminescence- or fluorescence-based. Autoradiography was the primary method for detecting horseradish peroxidase-conjugated (HRP), chemiluminescent signals. X-ray film can be quick and sensitive for qualitative purposes, but it entails the use of hazardous materials, a dark room, and a basic office scanner for densitometry analysis. For the quantitation of protein expression, the limited dynamic range associated with film and the manual scanning process pose accuracy challenges.1 A popular alternative approach are digital imaging stations equipped with a charge-coupled device (CCD) camera. CCD digital workstations have been optimized for increased sensitivity (i.e., photon-capturing ability) and dynamic range, resulting in an enhanced ability to detect differences in band density. This imaging modality, therefore, represents an upgraded alternative to autoradiography for precisely quantitating relative protein expression. However, chemiluminescent CCD imaging is best suited for single-protein studies. Where more than one protein is of interest, particularly if similar in size but notably different in abundance, fluorescent multiplexing with multi-channel fluorophore emission detection is superior for demonstrating quantitative differences.2
Imaging of multiple fluorophores is possible using both CCD and laser-based imagers. The choice between CCD and laser instruments is often governed by the need for chemiluminescence, colorimetric imaging, and densitometry, all ideal tasks for CCD-based imaging systems. For high spatial resolution, a broad linear detection range and many multiplexing options with a laser scanner are the best choices. State-of-the-art laser scanners are equipped with advanced detectors, including photomultiplier tubes that excel in detecting both weak and strong signals over a broad spectral range. Knowledge of an imaging modality’s linear detection range is essential to distinctly quantitating the expression level of multiple proteins of varying abundance. The linear detection range refers to the digitally-detected signal intensity (i.e., laddered bands) as a reflection of the actual protein quantity within a sample across all lanes on a single blot.3 Of the detection methods available, fluorescence-based imaging has the broadest linear detection range. Fluorescently labeled probes are not dependent on an enzyme-substrate reaction but are instead directly detected and have a quantifiable linear detection range ten times greater than that of chemiluminescent substrates.1 The dynamic range of film is the narrowest of the three, making it the least accurate choice for protein expression quantitation–particularly for relative measurements involving low abundance proteins.
Saturation, or overexposure, will occur if the protein’s chemiluminescent or fluorescent signal is not within the linear range of detection. Membrane saturation is commonly observed when imaging highly abundant proteins and can be corrected by manually reducing the sensitivity of the CCD or fluorescent camera (i.e., increasing resolution to require more signal for oversaturation).1 However, the chemiluminescent signal emitted is not always proportional to actual protein abundance and amplifying this further can be problematic. Sometimes, it is necessary to troubleshoot primary and secondary antibody concentrations to resolve saturation problems successfully.
The signal-to-noise ratio compares the amount of generated signal that is specific to the target protein versus the nonspecific background signal, or noise. Both fluorescently-tagged and HRP-conjugated secondary antibodies are common culprits of nonspecific binding.2 A high signal-to-noise ratio can be resolved by careful optimization of blocking conditions–including the chosen agent, concentration, and incubation time. The stripping and reprobing process that is characteristic of chemiluminescent-dependent imaging removes bound proteins unequally from the membrane, resulting in a varying range of epitope abundance and accessibility when reprobing with a new primary antibody. This discrepancy is perpetuated in the subsequent secondary antibody incubation step, where most of the nonspecific binding occurs.2 The nonspecific binding of HRP-conjugated secondary antibodies picks up a luminescent signal when exposed to substrate creating increasingly higher levels of background noise with each strip and reprobe performed.2 Fluorescence-based imaging, however, makes simultaneous detection of proteins possible and eliminates the need for a repeat multi-step detection procedure. Fluorescent-dependent detection methods are therefore not only time-saving options but are also more consistent.
Of the imaging modalities available, fluorescence has evolved to be a popular choice as an ultra-sensitive, flexible, and convenient approach for quantitative Western blotting. Even still, chemiluminescent CCD imaging continues to offer superb advantages over traditionally used autoradiography methods that are now appropriate for qualitative analyses only. Understanding the similarities and differences between each approach is central to recognizing when there is an advantage to one over another.
1. Bass JJ et al, “An overview of technical considerations for Western blotting applications to physiological research,” Scan J Med Sci Sports 27:4-25, 2016
2. Gingrich CJ, “Multiplex detection and quantitation of proteins on Western blots using fluorescent probes,” BioTechniques 29:636-642, 2000.
3. Posch A, Taylor CS, “The design of a quantitative western blot experiment,” Biomed Res Intl 2014:361590, 2014.