While a uniform background is relatively easy to subtract, noisy background may obscure a weak signal and is difficult to subtract from the analysis. This variation relates to the amount of noise that is accumulating within that signal. For example, a pixel may have a value of 1,000 during one instance, but a value of 1,100 the second time it is measured. Noise is the statistical uncertainty when measuring the intensity at a pixel. This background is often a result of incomplete blocking, insufficient washing of excess antibody, or an improperly hydrated PVDF membrane (when using fluorescence detection). The goal for researchers performing western blots is to maximize the band from the protein of interest while minimizing the background seen on the membrane. Understanding both will allow you to better evaluate an imaging system and any images taken with that system. Two important and related measures of an imaging system’s performance are: its ability to generate images with a high signal-to-background, and high signal-to-noise ratios. Selection of an imaging system is no different and the most sensitive systems can detect low signal while introducing low levels of background noise that may obscure detection of that signal. Optimization of the upstream steps of western blotting for maximum sensitivity is largely about maximizing signal while minimizing background. Signal, Background, and Noise in Western Blot Imaging In this example, the last two bands in this serial dilution are visible to the eye, even though the pixels making up the band are barely above background. Our eyes can discern even very faint bands above background given the knowledge of this size and shape. On a western blot, we are detecting bands that have an expected size and shape, rather than single pixels. In this example, even though system B shows a lower band intensity, it has superior signal to background so would be able to detect a fainter signal on the blot with a longer exposure time. For a single pixel, one common definition is the lowest intensity that can be identified with a 99% confidence (example, greater than or equal to three standard deviations of baseline noise.) The limit of detection is the lowest intensity that can be confidently identified within a background. This method provides a rough comparison of sensitivity, but if only intensity is evaluated and not measurements of signal to background or signal to noise, the system's limit of detection can be misestimated. Band intensity is often used to compare imaging systems. When evaluating candidate imaging systems for purchase, many researchers will take images of the same blot using identical (or near identical) settings. After all, the whole western blotting experiment can take upwards of two days so a few extra minutes during image acquisition is a small price to pay to see a low-expressing protein. It is important to consider the instrument with the lower ultimate limit of detection, as long as it takes a reasonable amount of time to acquire that image. While quick and easy to perform side-by-side, this method compares time to results, rather than the ultimate sensitivity of the imagers. Often, two imagers are evaluated by imaging the same blot with identical imaging times and comparing the resulting band intensities. In western blotting, sensitivity of imaging instruments is often discussed using two somewhat differing definitions. This requires optimization of all the upstream steps of the western blotting workflow as well as an imaging system that has the highest sensitivity possible. One of the most common challenges for a western blotting experiment is the detection of low-abundance protein targets.
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