Chemiluminescent Westerns: How Film And

Chemiluminescent Westerns: How film and photochemistry affect experimental results LI-COR® Biosciences • 4647 Superior Street, Lincoln, NE 68504 • www.licor.com Table of Contents Page 1. Introduction . 1 2. Photochemistry of x-ray film. 2 2.1 The photographic emulsion. 2 2.2 The Reciprocity Law and reciprocity failure . 3 Physics and statistics affect silver grain activation . 3 Faint signals: low-intensity reciprocity failure . 3 Strong signals: high-intensity reciprocity failure . 4 2.3 Image clarity and resolution . 5 “Blow-out” and spreading of strong signals . 5 Film handling and processing artifacts . 5 Parallax. 6 3. Types of x-ray film used for chemiluminescent Westerns . 6 Choosing an x-ray film . 6 4. Quantification of chemiluminescent Westerns . 6 4.1 Variables that affect quantification . 7 Detection chemistry (enzyme/substrate). 7 Signal capture and densitometry . 7 4.2 Densitometry. 8 Important factors in densitometry. 9 4.3 Examination of film response, using an LED light source . 9 5. Conclusions & 6. References . 11 1. Introduction Since the 1970s, enhanced chemiluminescence has been used to detect proteins on Western blots.1 Sec- ondary antibodies are labeled with the horseradish peroxidase (HRP) enzyme, which oxidizes the luminol- based chemiluminescent substrate and causes it to transiently produce light at ~428 nm (Fig. 1). This sensitive, reliable detection chemistry is typically documented by exposure of the blot to x-ray film, which darkens in response to the emitted light. Signal intensity is determined by the number of HRP molecules reacting with substrate. Chemiluminescent blots are often analyzed by visual assessment of band intensities. This method is sufficient to confirm presence or absence of a signal, or to compare bands of substan- tially different intensities. For more detailed analysis, film images may be digitized and relative band intensities measured by densitometry. Figure 1. Enhanced chemiluminescence. Luminol is a widely used chemiluminescent reagent. Oxidation of luminol by peroxide creates an excited-state product, 3-aminophthalate. Photons of light are transiently produced when this product decays to a lower energy state. Chemiluminescent Westerns: How film and photochemistry affect experimental results – Page 2 Although most researchers have used film to document Western blots, many may be unfamiliar with the photochemical process that creates a visible image on a sheet of x-ray film. Because this process affects data output, it is important to understand how chemiluminescent signals are recorded by film – particu- larly if the results will be quantified by densitometry.2 This paper describes the effects of photochemistry on the response of film to both faint and strong signals, image quality, image clarity, and quantification by densitometry. The accuracy of densitometry depends on the sensitivity, linear response range, and exposure time of the film. 2. Photochemistry of x-ray film 2.1 The photographic emulsion Photographic emulsions were first introduced in the 1850s, and the basic principles remain unchanged. The photographic film used to document chemiluminescent Western blots is coated with an emulsion that contains light-sensitive silver halide crystals (also called “grains”). • Photons of light activate the silver grains, converting some silver ions to silver atoms. This creates a latent image. • During film processing, the silver atoms in each activated grain catalyze the reduction of the entire grain to black metallic silver. This creates the visible image on film. • Unexposed silver crystals are dissolved and washed away during processing. The concentration of metallic silver that remains on the film after development is called optical density (OD). The degree of darkening is related to the intensity of light exposure. OD and densitometry are discussed further in Section 4. The “characteristic curve” demonstrates the response of a film to the full range of possible exposures (Fig. 2).3 The sigmoidal, non-linear shape of this curve is caused by the physics and statistics of silver grain activation, which are discussed in detail below. Figure 2. Characteristic curve of X-ray film (adapted from Kodak, 2007). The “toe” region of the curve indicates very low exposures. The center or “straight line” region is the approximate linear response range, where OD is proportional to the log of the exposure. The “shoulder” region represents higher exposures and is relatively flat. The slope of the “straight line” region indicates the film’s scale of contrast. A steeper curve indicates a shorter scale of contrast and narrower dynamic range. LI-COR Biosciences www.licor.com Chemiluminescent Westerns: How film and photochemistry affect experimental results – Page 3 2.2 The reciprocity law and reciprocity failure: Physics and statistics affect silver grain activation The response of film to light is governed by the reciprocity law. This law states that film response is determined by total exposure, which is dependent on light intensity and exposure time: light intensity x duration = total exposure The reciprocity effect is an inverse relationship. Bright light delivered for a shorter duration can produce the same response as dim light delivered for a longer duration. This holds true across a range of values, but becomes inaccurate outside that range. When signals are very strong or very faint, the relationship falls apart and film response is no longer proportional to light intensity and duration (Fig. 2). This is called reciprocity failure. It occurs at both high and low intensities of light, but with different mechanisms.4 Faint signals: low-intensity reciprocity failure At very low intensities of light, film is less responsive and is therefore disproportionately insensitive. Although this property keeps background low, it also causes faint signals to be under-represented.4 The response of film to light is affected by the rate at which photons are received. If the film receives 100 photons of light all at once, it will react and create a latent image; however, if 100 photons filter in slowly over one hour, they will probably not be detected. This non-linear response is caused by the physics of silver crystal activation (described below).2,4 A minimum threshold level of exposure is required to expose a silver halide crystal and create a latent image. When a crystal is activated by one or two photons of light, it is unstable and rapidly reverts back to its stable, inactive form. Multiple photons must be absorbed by the activated silver crystal before it reverts, to form a stable latent image that can be developed during film processing. When signals are faint and require long exposures, a stable latent image is unlikely to form. As a result, faint signals are underestimated and appear to drop off rapidly on film. This is often observed in dilution series of samples, when it seems to the eye that additional faint bands should be visible (Fig. 3). However, signals below a certain intensity level simply cannot be detected, even with extremely long exposures. Reciprocity failure effects can be reduced by pre-exposure of the film to an instantaneous flash of light. This approach to hypersensitization of film was introduced in the mid-20th century.5 “Pre-flashing” by- passes the reversible stage of latent image formation, such that multiple photons are not required to fully activate each silver grain.4 This increases sensitivity and creates a more linear relationship between OD and light exposure for low-intensity signals; however, pre-flashing is inconvenient and difficult to perform reproducibly. A) B) Figure 3. On film, faint signals are underestimated and drop off very quickly. ERK2 was detected in serially diluted NIH/3T3 cell lysates. A) SuperSignal® West Pico substrate and 5-min film exposure. B) Custom ECL substrate and 5-min film exposure. On both blots, signal drops off abruptly even though it seems that additional bands in the dilution series should be visible. LI-COR Biosciences www.licor.com Chemiluminescent Westerns: How film and photochemistry affect experimental results – Page 4 Strong signals: high-intensity reciprocity failure The non-linear response of film to strong signals is caused by the statistics of silver grain activation. Most researchers are aware that strong signals cause saturation of film, a point at which all silver grains have been activated. Once saturation is reached, no further signals can be recorded, regardless of their intensity. However, it is also important to recognize that film response begins to plateau well before saturation is reached. As the film becomes progressively more exposed and more silver grains are activated, each new photon of light is statistically less likely to strike an unactivated grain.2,4 This under-represents strong signals, causing a logarithmic, non-linear response in darkening and OD prior to saturation (Fig. 4). As strong signals approach the maximum possible density, they lose their ability to show tonal variations on the developed film. This “overexposure” causes high- and moderate-intensity bands to appear similarly dark and dense. The non-linear response of film to strong signals contributes to its narrow linear range.4 Figure 4. Strong signals plateau and become saturated. Akt was detected in serial dilutions of NIH-3T3 cell lysate, using SuperSignal® West Dura substrate and 5-min film exposure. Strong signals are underestimated (boxed region); they plateau and fail to show tonal variation. Densitometry clearly shows the lack of linear response at higher con- centrations. The linear range in this experiment spans only 3-4 dilutions (4- to 8-fold). The tendency of strong bands to blur and spread on film (“blow-out”) also makes quantification difficult. Stronger bands do not have clear margins; they obscure adjacent bands and cannot be accurately sepa- rated (Fig. 5). This is especially problematic when stronger bands are located near faint bands, because the longer exposures required for detection of faint bands increase the spreading of stronger bands.

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