Chemiluminescent Westerns:

How lm and photochemistry

affect experimental results
LI-COR

Biosciences 4647 Superior Street, Lincoln, NE 68504 www.licor.com

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 lm, 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 intensi-
ties. This method is sufcient to conrm presence or absence of a signal, or to compare bands of substan-
tially different intensities. For more detailed analysis, lm images may be digitized and relative band
intensities measured by densitometry.
Table of Contents Page
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Photochemistry of x-ray lm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 lm used for chemiluminescent Westerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Choosing an x-ray lm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4. Quantication of chemiluminescent Westerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4.1 Variables that affect quantication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Detection chemistry (enzyme/substrate). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Signal capture and densitometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.2 Densitometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Important factors in densitometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
4.3 Examination of lm response, using an LED light source . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5. Conclusions & 6. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
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 tran-
siently produced when this product decays to a lower energy state.
Although most researchers have used lm to document Western blots, many may be unfamiliar with the
photochemical process that creates a visible image on a sheet of x-ray lm. Because this process affects
data output, it is important to understand how chemiluminescent signals are recorded by lm particu-
larly if the results will be quantied by densitometry.
2
This paper describes the effects of photochemistry
on the response of lm to both faint and strong signals, image quality, image clarity, and quantication
by densitometry.
2. Photochemistry of x-ray lm
2.1 The photographic emulsion
Photographic emulsions were rst introduced in the 1850s, and the basic principles remain unchanged.
The photographic lm 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 lm 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 lm.
Unexposed silver crystals are dissolved and washed away during processing.
The concentration of metallic silver that remains on the lm 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 lm to the full range of pos-
sible exposures (Fig. 2).
3
The sigmoidal, non-linear shape of this curve is caused by the physics and statis-
tics of silver grain activation, which are discussed in detail below.
The accuracy of densitometry depends on the sensitivity,
linear response range, and exposure time of the lm.
Figure 2. Characteristic curve of X-ray lm (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 rep-
resents higher exposures and is relatively at. The slope of the straight
line region indicates the lms scale of contrast. A steeper curve indi-
cates a shorter scale of contrast and narrower dynamic range.
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2.2 The reciprocity law and reciprocity failure:
Physics and statistics affect silver grain activation
The response of lm to light is governed by the reciprocity law. This law states that lm response is deter-
mined 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 lm 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, lm 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 lm to light is affected by the rate at which photons are received. If the lm receives 100 pho-
tons of light all at once, it will react and create a latent image; however, if 100 photons lter 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 re-
verts, to form a stable latent image that can be developed during lm processing. When signals are faint
and require long exposures, a stable latent image is unlikely to form. As a result, faint signals are under-
estimated and appear to drop off rapidly on lm. 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 lm to an instantaneous ash of light.
This approach to hypersensitization of lm was introduced in the mid-20th century.
5
Pre-ashing 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-ashing is inconvenient and difcult to perform
reproducibly.
Figure 3. On lm, faint signals are underestimated and drop off very quickly. ERK2 was de-
tected in serially diluted NIH/3T3 cell lysates. A) SuperSignal

West Pico substrate and 5-min

lm exposure. B) Custom ECL substrate and 5-min lm exposure. On both blots, signal
drops off abruptly even though it seems that additional bands in the dilution series should
be visible.
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A)
B)
Strong signals: high-intensity reciprocity failure
The non-linear response of lm to strong signals is caused by the statistics of silver grain activation. Most
researchers are aware that strong signals cause saturation of lm, 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 lm response begins to plateau well before saturation is
reached. As the lm 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 sig-
nals, 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 de-
veloped lm. This overexposure causes high- and moderate-intensity bands to appear similarly dark and
dense. The non-linear response of lm to strong signals contributes to its narrow linear range.
4
The tendency of strong bands to blur and spread on lm (blow-out) also makes quantication difcult.
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.
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 lm 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).
Figure 5. On lm, strong signals blur and
spread to obscure adjacent bands. ARNO
protein was detected in cell lysates, using
ECL Plus substrate and 90-sec lm exposure.
Spreading of bands is especially problematic
when longer exposures are required to de-
tect faint bands (right side of blot).
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The nuances of photochemistry introduce variability
into Western blot analysis.
The nuances of photochemistry introduce variability into Western blot analysis. Optical density (OD) is not
a direct function of light generation by the chemiluminescent substrate. It is an indirect function that de-
pends on the response of the lm to light.
2
The accuracy of densitometry depends on the sensitivity, linear
response range, and exposure time of the lm. Reciprocity failure introduces error and limits the ability of
lm to present a complete picture of the data. To complicate matters further, reciprocity failure can occur
differently at different locations on the photographic emulsion.
2.3 Image clarity and resolution
When chemiluminescent Western blots are documented with lm, band resolution and clarity are often
compromised. Several key factors are involved.
Blow-out and spreading of strong signals. On lm, strong signals blur and spread obscuring adjacent
bands (Fig. 5). If you are attempting to detect both strong and faint signals on the same blot, or several
bands in close proximity, blown-out bands can be a serious limitation (Fig. 6).
Blurring and spreading of bands is primarily caused by scattering of light. During lm exposure, light is
simultaneously collected from all areas of the blot. As light scatters, it travels away from the site where it
originated and activates silver grains in the surrounding area. This creates large, diffuse bands that expand
in size and frequently obscure adjacent bands. Longer lm exposures increase the severity of blurring and
spreading. In addition to light-scattering issues, the layer of liquid substrate, plastic wrap, and the lm
itself can act as waveguides to encourage horizontal propagation of light. This phenomenon further pro-
motes spreading of bands.
Film handling and processing artifacts. Image quality is also affected by artifacts that occur during expo-
sure and processing. If the blot or lm shifts during handling, blurry bands and after-images may occur,
especially if signals are strong. Chemiluminescent blots must be wrapped in plastic before exposure to
lm, to contain the liquid substrate. Leakage of liquid during exposure will cause artifacts on the devel-
oped lm, as will static electricity from the plastic wrap. During processing, scratching and mechanical
damage may occur (Fig. 7). If the sheet of lm becomes caught inside the automatic developer, scratching
can be severe.
Figure 6. Bands that are close together may
not be clearly resolved. The ERK1/ERK2 dou-
blets in these A431 cell lysates cannot be sep-
arated and resolved at higher protein concen-
trations, even in this short, 15-second exposure.
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Figure 7. Scratches and other artifacts affect image quality on
lm. In this 30-sec exposure, scratches and static electricity
artifacts are observed.
Parallax. Most commercial X-ray lm is double-emulsion (light-sensitive emulsion coatings are applied to
both sides of the lm). This provides maximum sensitivity and ease of use, but reduces image clarity and
sharpness. When a Western blot is exposed to double-emulsion lm, images are created on both emul-
sions (both sides of the lm), then superimposed. This enhances sensitivity, but causes an unwanted paral-
lax effect. Because the two images have different path lengths, the image on the far side of the lm is
slightly larger than the image on the near side. When the two images are superimposed, the parallax effect
causes blurring of the edges and reduces the sharpness of the resulting bands.
6
If single-emulsion lm is
used to avoid parallax, detection sensitivity will be reduced.
3. Types of X-ray lm used for chemiluminescent Westerns
Many brands and varieties of X-ray lm are available for documentation of chemiluminescent Westerns.
Commercially available X-ray lms have many similarities. Most lms:
Use a polyester base to support the light-sensitive photographic emulsion, with an outer layer of
supercoat material to protect the emulsion from scratching, mechanical damage, and static.
Are double-emulsion (the lm is coated with photographic emulsion and anti-scratch material on
both sides). This increases lm speed and sensitivity, but may cause blurring due to parallax.
Are sensitive to blue and green light (orthochromatic). They are not sensitive to red light and are
compatible with red darkroom safelights, although safelights may cause fogging.
7
The polyester support layer is typically grey or blue in color. Blue-tinted lm has been used for many
years. The blue color is believed to reduce glare and eyestrain during transillumination, and may improve
visualization.
7
Some newer, high-performance lms are made with a clear base that is thought to improve
visualization of very faint bands.
Choosing an X-ray lm. Proper lm choice is determined by the speed (sensitivity) and resolution re-
quired. Speed and resolution are determined by the size of the silver halide crystals and thickness of the
photographic emulsion. Larger crystals and thicker emulsions enhance sensitivity, but at the expense of
resolution. Finer crystals and thinner layers provide greater detail. Maximum-resolution lms are best
suited to lower energy -emitters such as
33
P and
14
C. High-performance lms such as BioMax Light and
Hyperlm ECL are recommended for chemiluminescence and high-energy emitters such as
32
P.
Film sensitivity varies widely, and high-performance lms are considerably more expensive. Price varies
widely, ranging from ~ $0.50 to $10 per sheet. Individually-wrapped lms are much more expensive than
those in standard, alternate-interleaved packaging. Blue-tinted lm is typically more economical but some-
what less sensitive. For maximum performance, a more expensive grey- or clear-base lm may be pre-
ferred. Because limited technical specications are provided, it is very difcult to compare different lm
products without side-by-side testing.
4. Quantication of chemiluminescent signals
Chemiluminescent Western blots are sometimes used for quantication. Densitometry of exposed lms is
a common approach. The accuracy and dynamic range of this method are variable, and proper controls
are critical for data interpretation.
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4.1 Variables that inuence quantication
Two important variables affect accuracy when chemiluminescent Western blots are quantied.
Detection chemistry (enzyme/substrate kinetics). Chemiluminescence is chemical reaction that releases
energy in the form of light. Enzymatic amplication of signal provides high sensitivity, but signal intensity
is dynamic and transient. Intensity peaks shortly after substrate is applied, and then fades exponentially
with time as the chemical reaction slows. Persistence of signal depends on the chemiluminescent sub-
strate used. Extended-stability substrates are typically more expensive than substrates that fade quickly.
Substrate availability and exhaustion are also signicant concerns.
1
Substrate distribution and accessi-
bility are never uniform across the entire surface of the blot. Unevenly distributed substrate, pooled sub-
strate, and bubbles can affect substrate availability and cause localized variation in signal intensity (shown
in Fig. 8). Because enzyme/substrate kinetics govern the generation of signal, intensity is unlikely to be
proportional to the amount of target present, or its proportionality may change as the reaction proceeds.
Strong bands with high concentrations of HRP enzyme may cause rapid local depletion of the substrate,
generating central white-outs in strong bands where light can no longer be generated.
The limitations of enzymatic detection are intrinsic to chemiluminescent Westerns, regardless of the signal
capture method used. Non-enzymatic detection, such as uorescent detection with uorophore-labeled
secondary antibodies, eliminates this source of variability.
Signal capture and densitometry. Chemiluminescent signals are often captured by exposure to X-ray lm.
This method is very sensitive, because long exposures can be used to detect low-intensity signals; how-
ever, the response of lm to light intensity is non-linear. Because of reciprocity failure, strong and faint
signals may not be accurately captured and the range of linear response is quite narrow.
4
Several lm exposures of different lengths are typically required to capture the desired image. Exposure
time must be long enough to detect the desired target, but short enough to limit undesired background.
Figure 8. Substrate availability can affect signal intensity on chemiluminescent Westerns. The same pair of
samples was loaded three times on a single blot (10 g or 5 g of C32 cell lysate; circled areas). SuperSig-
nal West Pico substrate was used for detection, with 5 min lm exposure. Non-uniform distribution of sub-
strate across the blot made some signals noticeably stronger than others (orange circle).
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A single exposure can only provide optimal detection across a narrow range of signal intensities. If the
exposure is too long, background may begin to obscure the signal. Stronger bands merge and blur to-
gether, and blown-out bands make quantication difcult. Reciprocity failure causes faint signals and
strong signals to be under-represented (Fig. 9). Saturation prevents measurement of further change in
signal intensity, and prevents accurate comparison of band intensities.
4.2 Densitometry
After lm exposure, densitometry is sometimes used to quantify protein levels on Western blots. This
method measures the optical density (OD), or degree of darkness, of photographic material. It is important
to remember that OD is not a direct function of light generation by the chemiluminescent substrate.
2
It is
an indirect function that depends on the response of lm to light, which is non-linear. The accuracy of den-
sitometry is therefore dependent on the sensitivity, linear response range, and exposure time of the lm.
Densitometry measures the degree of darkness of the image as a function of light transmission; density
equals the logarithm of the reciprocal of transmittance. For example, if a region transmits one-tenth of the
incident light, its density is equal to 1 (Table I).
10
Figure 9. Densitometry of lm is limited by signal saturation. A) Puried AKT was detected on a Western blot
with chemiluminescence and exposure to lm. B) Signals were quantied by densitometry of the exposed lm.
The sigmoid curve indicates signal saturation and limited dynamic range using densitometry and lm.
Reprinted from Wang et al, 2011.9
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OD is not a direct function of light generation by the
chemiluminescent substrate. It is an indirect function that
depends on the response of lm to light, which is non-linear.
% Light Transmitted Optical Density (OD)
80% 0.1
60% 0.2
40% 0.4
20% 0.7
10% 1.0
5% 1.3
1% 2.0
Table I. Relationship between light transmittance and OD.
10
A) B)
For Western blot analysis, the useful range of densities is approximately 0.2 2.0 (roughly 10-fold).
2
When
unexposed lm is processed, it has an OD of 0.15 - 0.20. This is called the gross fog density of the lm, and
signals must exceed this density to be recorded. An OD of 1.0 indicates a medium grey tone (halfway be-
tween white and black). High ODs (above 2.0) appear black and indicate saturation; they cannot be discrim-
inated from one another. The overall response of lm is non-linear, but approaches linearity over narrow
ranges (Fig. 9). A standard curve is strongly recommended to conrm the linear range of lm
response for each experiment.
Important factors in densitometry
The variable nature of Western blot densitometry methods was examined in detail by Gassmann et al
(2009).
11
This study correlated plasma Epo (erythropoietin) values for various mammalian species, deter-
mined by radioimmunoassay (RIA) and Western blot densitometry. Densitometry was performed under
28 different conditions. Variables included method of image acquisition (ofce scanner or CCD system),
background correction, software, and denition of OD value. The comparison was based on the p-value
of the linear regression between RIA and Western blot values. Depending on the densitometry conditions
used, the p-value of the regression line ranged from 0.000013 to 0.76. The same data could be used to
make statistically valid, but completely contradictory statements about the correlation between RIA and
Western blot results.
This 2009 study describes two key pitfalls in densitometry.
11
First, variability can arise during digitization of lms. Ofce scanners are frequently used, but may
provide limited dynamic range and uneven illumination of the scan area. More importantly, many
desktop scanners have automatic gain controls that make the OD of a band also a function of the
surrounding image structure. Automatic gain control resulted in 1.5- to 105-fold variation in the
measured OD of identical spots, due to differences in the structure of the rest of the image. Auto-
matic gain controls should not be used when scanning lms for densitometry.
The second pitfall involves the method of measurement. Important factors include the background
correction algorithm, the sample tool size relative to lane width, and the denition of OD value (the
integral or sum of all peak heights is recommended). The authors consider image acquisition and
software parameters to be an important aspect of Western blot densitometry and analysis.
4.3 Examination of lm response using an LED light source
To examine signal capture issues and reciprocity failure in more detail, it is helpful to use a model system
that eliminates all variability contributed by detection chemistry. One approach is to replace the enzyme/
substrate reaction with a light-generating LED performance test target (PTT) manifold.
12
The PTT emits
calibrated light across a wide range of intensities, using an LED backlight panel with regulated brightness
levels that decrease in two-fold steps (Fig. 10). There are four duplicate slots for each brightness level, and
a total of 18 brightness levels. The PTT is ideal for direct testing of lm response because detection chem-
istry is eliminated, creating consistent and reproducible light intensities that are unaffected by timing.
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The PTT manifold was used to generate a series of lm exposures (Fig. 11) that were analyzed by densito-
metry.
12
High-intensity reciprocity failure was clearly observed. As strong signals caused lm to become
progressively more exposed, the optical density of bands began to plateau. Because each new photon of
light was statistically less likely to strike an unactivated silver grain, strong signals were under-represented
on lm. Near the saturation point, bands no longer showed tonal variations on the developed lm. Over-
exposure caused strong and moderate bands to appear similarly dark and dense (Fig. 11A).
Quantication of signals from the PTT manifold is shown in Fig. 12. All exposure times (1 sec - 15 min)
demonstrated plateau and saturation of strong signals even when exposure time was limited to 1 sec.
Each exposure displays a narrow range of linear response (4 - 8-fold), but the boundaries of that range
are different for each exposure. This illustrates the importance of using an appropriate standard curve for
analysis of protein levels with lm and densitometry.
Figure 10. Layout of the PTT light manifold. The
LED backlight panel emits regulated levels of
brightness, decreasing in two-fold steps. There
are four duplicate slots for each brightness level.
Each slot is 8 mm x 1 mm. Slots are spaced 1 mm
apart within each brightness level, and 1.5 mm
from one level to another.
Figure 11. Saturation of strong signals on lm. A) 15-second lm exposure of PTT light manifold. Strong and mod-
erate signals appeared similarly dark and dense. The strongest signals (top left) were saturated, and had begun to
blur and spread. Weaker signals (A, right) were not visible in this short lm exposure. B) 1-minute lm exposure.
All strong bands appeared dark and dense. The strongest signals (B left) showed signicant blurring. Weaker sig-
nals (B, right) began to be detected. C) 15-minute lm exposure. This long exposure allowed weaker signals to be
detected (C, right), but those began to saturate and blur at the top. Strong signals (B, left) were saturated and
blurred together completely.
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One brightness level
5. Conclusions
Exposure to lm is a sensitive and widely-accepted method for documentation of chemiluminescent West-
ern blots. However, the photochemistry of lm has several inherent limitations that affect data output and
can introduce error. These include the non-linear response of lm to faint and strong signals (reciprocity
failure), lack of image clarity caused by blurring and spreading of bands, and the narrow range of optical
densities that can be recorded by lm.
These limitations may affect interpretation of Western blot data, but are not commonly acknowledged or
discussed. For experiments that require precise quantication of protein levels on Western blots, an alter-
native detection method may be appropriate.
6. References
1. Whitehead, TP, LJ Kricka, TJN Carter, and GHG Thorpe. Analytical luminescence: its potential in the
clinical laboratory. Clin Chem. 25(9): 1531-46 (1979).
2. Baskin, DG and WL Stahl. Fundamentals of quantitative autoradiography by computer densitometry
for in situ hybridization, with emphasis on
33
P. Journal of Histochemistry and Cytochemistry
41(12):1767-76 (1993).
3. Kodak. The essential reference guide for lmmakers: Basic sensitometry and characteristics of lm.
Kodak Educational Products, Code: H-845 CAT No. 145 6144. Eastman Kodak Company (2007).
4. Laskey, R.A. Efcient detection of biomolecules by autoradiography, uorography or chemilumines-
cence. Methods of detecting biomolecules by autoradiography, uorography and chemiluminescence.
Amersham Life Sci. Review 23:Part II (1993).
5. Bowen, IS and LT Clark. Hypersensitization and reciprocity failure of photographic plates. J Optical
Society Amer. 30:508-10 (1940).
6. Kopans, DB. Breast imaging, 3rd edition. Lippincott Williams & Wilkins (2007).
7. Dickerson, RE. Discussion of fog standards for general-purpose radiographic lm. Carestream Health.
www.carestream.com/Fog_Standards--General_Purpose_Radiographic_Films
Figure 12. Quantitative analysis demonstrates under-representation and saturation of strong signals. The PTT
manifold was used to perform a series of lm exposures (1 sec, 5 sec, 15 sec, 1 min, and 15 min). Films were
analyzed by densitometry. At all exposure times, strong signals quickly reached a plateau. The log/log plot (right)
illustrates the narrow linear range of lm detection (<10-fold). Light intensity values that fall within the linear
range are different for each exposure.
Chemiluminescent Westerns: How lm and photochemistry affect experimental results Page 11
LI-COR Biosciences www.licor.com
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interfering RNA suppression demonstrate that plasminogen activator inhibitor-1 produces elevated
collagen accumulation in normal and keloid broblasts. American Journal of Pathology 173(5):1311-25
(2008).
9. Wang, X, Y Dong, AJ Jiwani, Y Zou, J Pastor, M Kuro-o, AA Habib, M Ruan, DA Boothman, and
C-R Yang. Improved protein arrays for quantitative systems analysis of the dynamics of signaling
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10. Davis, R and FM Walters Jr. Sensitometry of photographic emulsions and a survey of the characteris-
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12. Anderson, JP et al. Evaluation of signal capture methods with custom-built LED backlight manifold
(performance test target; PTT). LI-COR Biosciences, unpublished data (2012).
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