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Film and CCD Imaging of Western Blots: Exposure time, signal saturation, and linear dynamic range Table of Contents Page 1. Introduction ..............................................................1 2. Experimental Design .......................................................2 3. Materials and Methods .....................................................3 4. Results and Analysis .......................................................3 4.1. Linear dynamic range, sensitivity, and limit of detection (LOD) ................3 4.2. Experimental linear dynamic range is more limited than theoretical dynamic range. .6 4.3. Impact of exposure time on linear dynamic range . ...........8 4.4. Signal-to-noise analysis. .10 5. Discussion ..............................................................12 5.1 Choosing an appropriate detection method ..............................12 5.2 Impact of signal saturation and lower limit of detection .....................13 5.3 Enhancing Western blot reproducibility .................................13 6. References ..............................................................15 1. Introduction Linear dynamic range is a critical factor in quantitative analysis of Western blot data. In the cell, en- dogenous protein levels span a vast dynamic range (an estimated 4-10 orders of magnitude).1 Because protein levels vary so widely, accurate Western blot analysis requires detection methods that can accommodate a wide dynamic range and effectively capture the richness and complexity of the data. Many common detection methods cite a wide theoretical linear dynamic range. In practice, howev- er, the available linear dynamic range may be much narrower and is generally limited by saturation of strong signals. Multiple exposures are usually captured for each blot, in an effort to mitigate the impact of signal saturation and determine the optimal exposure time with the widest linear dynamic range. But this practice can affect the reproducibility and reliability of data, because the chemiluminescent reaction is not constant and light output changes over time. It is important to identify and explore factors that influence linear dynamic range, to understand and minimize their impact on data analysis and interpre- tation. Linear dynamic range is the span of signal intensities, from faintest to strongest, that displays a linear relationship between light emission from the sample and signal intensity recorded by the detector. This study investigates the linear dynamic range and limit of detection for several common Western blot Page 2 – Film and CCD Imaging of Western Blots: Exposure time, signal saturation, and linear dynamic range detection methods. Film exposure, a conventional charge-coupled device (CCD) imaging system (Imager B), and an alternative CCD system (Odyssey® Fc Imager) were tested and compared. Linear dynamic range, detection sensitivity, lower limit of detection (LOD), and signal-to-noise ratios (SNR) were examined. The relationships between exposure time, signal saturation, and linear dynamic range were also explored. 2. Experimental Design The goal of this study was to compare the limit of detection and linear dynamic range of imaging technologies commonly used for chemiluminescent Western blots. Two different CCD imaging systems and film exposure were chosen for testing. To ensure unbiased data interpretation, we sought to minimize uncontrolled variables that com- monly affect data acquisition. The most disruptive variable in chemiluminescent Western blotting is the kinetic, unstable nature of the detection chemistry. Chemiluminescent signals are governed by enzyme-substrate kinetics and are highly dependent on timing. To achieve maximum linear dynamic affected by mechanical shock and vibration, accidental exposure to light, temperature variation and aging. range, most detection methods require comparisonOther of factors multiple can affect the exposuresreading of luminometer tos suchdetermine as, lint, dirt or liquid the fumes optimal collected by the optic, liquid splashed on the optic, and mechanical or optical misalignment of the reading mechanism. 2 exposure time for each experiment. Because the Inintensity most application, of especially chemiluminescent in clinical applications, it is very signals important to verifychanges the performan ce of luminometers, because faulty readings can result in misdiagnosis of patients. over time, each exposure represents a unique stage in the kinetics of the enzyme/substrate reaction Good Laboratory Practice (GLPs) and many regulatory agencies, laws (such as CLIA 88) require the that cannot be recaptured. In this study, we removedluminometers this be variable periodically checked by tousing ensure that a the calibrated machine works as perluminometer factory specs. reference plate (Harta Instruments, Fig. 1) as a stableQuite often light when thesource. reading of a certain samples are not correct, the lab personnel automatically blames the luminometer, and they ship the machine back to the manufacturer, when the problem is with the reagent, (bad reagent, wrong temperature, contamination, etc), the process of preparing the reagent, or any myriad of problems associated with anything but the machine. This causes a lot of unnecessary The Harta reference plate is a NIST-traceable standardexpenses andwidely interruption used of service, to when verify all that needsand to bevalidate done is to read the a reference sen plate- to verify the performance of the luminometer. 3 sitivity, linearity, and dynamic range of luminometers and other imaging instrumentation. Highly The Reference plate has dual redundancy light sources, so one light source can be compared against the stable LEDs and a linear optical attenuator systemother. generate The reference platemultiple has a built inlight battery cpointsheck system. acrAs longoss as the seven battery check orders light is ON, the batteries are OK. These are provided to check the performance of the reference plate itself. When the of magnitude. Reference wells that emit very low batterylevels goes belowof light a safe level, enable well A8 will evaluation turn off to indicate weakof sensitivitybattery. and limit of detection (LOD). With the Harta device, weThe isolatedHarta RM-168-96 and luminometer examined reference plate the can beperformance used with any standard 96of well each microtiterplate luminometers. detection system in the absence of other experimental variables. These results reflect the intrinsic capabilities and limitations of each detection system,Since 1999, rather the Harta thanluminometer limitations reference plate h asof been the used Westernby thousands of satisfiedblot customers in over 25 countries worldwide. technique or detection chemistry. BOTTOM VIEW OF THE REFERENCE PLATE. Top view Bottom view Figure 1. Luminometer reference microplate. The Harta RM-168 device uses stable LEDs and a linear optical attenuator NISTsystem Traceable to generate Luminometer stable, Reference reproducible Microplate: light output across seven orders of magnitude. Light wavelength is 540 nm, and long- term stability is 5%. Features: • NIST traceable • CE compliant • 7 decades of stable light sources, checks: o Accuracy o Sensitivity o Dynamic Range o Linearity o Stability / repeatability -18 LI-COR Biosciences• Lowest level of light is equivalent to approximately 10 moles of luciferase using Promega www.licor.com/bio Bright-GloTM luciferase assay • Works in any standard 96 well luminometer • Dual redundancy light sources for the plate’s own performance verification • Built-in battery check plus weak battery indicator • Extended life lithium batteries • On-Off switch • Robust CNC machined aircraft grade aluminum construction • Supplied with padded hard storage case, spare battery pack, 2 switching tools, screwdriver and spare screws for the battery compartment • All electronics / optics system, no radioactive materials used in the plate The performance of a luminometer is largely dependent on the performance of a very delicate Photomultiplier Tube (PMT). The PMT which is actually an old vacuum tube technology device, is easily Page 3 –Film and CCD Imaging of Western Blots: Exposure time, signal saturation, and linear dynamic range 3. Materials and Methods A Harta RM168-96 luminometer reference microplate was used as the light source for all experi- ments (recommended recalibration date 1/15/15). The plate’s secondary, independent light source was used to verify light output and battery strength. The reference plate was imaged with X-ray film, a conventional CCD imager (identified here as Imager B), and an Odyssey® Fc dual-mode imager (LI-COR Biosciences). Signals were quantified and background adjustment was applied. Film: The Harta plate was used to generate a series of film exposures. In a darkroom, the plate was placed face-down onto Blue Lite autorad film (GeneMate). For each exposure, the light source was activated for the indicated time. Film was developed by standard procedures. Film images were digitized, and densitometry was used to analyze signal intensities. Upper limit of detection was inferred by densitometry, as indicated by loss of linear response (plateau and saturation) for stronger signals. Imager B: A conventional, commercially-available CCD imager was used. The Harta plate was placed face-up inside the imager, and activated. Various exposures were then captured by man- ual adjustment of image acquisition settings. Default exposure times did not produce acceptable results, so exposure times

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