Image analysis software typically offer tools to subtract background, depending on the method of band quantification used.Īn intuitive way to think about background is to think about how we measure the size of mountains. For situations where the background intensity is near that of the signal, or where the background is uneven, identifying which pixels belong to the band of interest, and identifying the relevant background to subtract can be challenging. This background can come from non-specific binding during antibody incubation, membrane background in the case of fluorescent images, or background arising from the instrumentation itself.įor bright, isolated bands on smooth backgrounds, this process can be relatively straightforward. No western blot is perfect, and all blot images show some level of background. Therefore, band quantity is often referred to as band "volume" by imaging software. Each pixel also carries signal intensity information, which can be thought of as data in the third dimension. The pixels that comprise the part of a band have x and y coordinates, so outline an area. Therefore, band quantity can be thought of as a volume, which is the total amount of signal for all the pixels within that band. Each pixel in a blot image has an x and y coordinate, in addition to an intensity value, which occupies the third dimension. A digital image of a blot can be thought of as data in three dimensions. A western blot image is made up of pixels, which contain information about how much signal was collected at each location in the image. Band VolumeĪ protein band is a feature that appears in a western blot image. Note: western blots are semi-quantitative so they are ideal for showing relative protein expression rather than an absolute quantity. By analyzing the intensity of the signal, you may determine whether the expression of the target protein in one sample has increased or decreased relative to another sample or control. The signal intensity of the band is directly proportional to the concentration of your target protein. Quantitating a western blot refers to the measurement of the signal emitted by your protein band(s) of interest. Blot Quantitation and Background What Does Quantitation Mean?
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