In addition, proteins of similar molecular weights can be detected concurrently providing enormous benefit when studying post-translational modifications such as phosphorylation. Loading and normalization controls can be detected on the same blot as the protein of interest. Fluorescent Westerns are ideal for detecting more than one protein simultaneously (multiplexing). In addition to documenting chemiluminescent Westerns, digital imagers also have the added capability of imaging fluorescent Western blots. Digital imagers also allow the convenience of digital records for storing and archiving experimental data.ĭigital imagers can be used for fluorescent Westerns Images can be manipulated immediately without waiting for a film to develop. The blot is placed in the digital imager unit and the signal is captured. Therefore a signal that may be saturating when captured on x-ray film may still be in the linear dynamic range when using a digital imager. Typically, a digital imager can record from 4,096-65,536 levels of intensity (approximately 2-4 orders of magnitude greater than x-ray film). CCD cameras detect chemiluminescent signals over a much broader range than film. Binning raises the amount of light collected in the pixel area, which increases the size of each pixel.įor ECL detection, CCD cameras are coupled with digital imaging systems to capture, document and analyze Western blots. In addition, detection sensitivity is increased by “binning” or combining the pixels. High-performance CCD cameras are capably of matching the speed and sensitivity of x-ray film by cooling the silicon chip to sub-zero temperatures, which reduces background noise. Costs for hazardous waste disposal of developing chemicals also increases the cost of using film.Ĭharge-coupled Devices and Digital ImagingĬharge-coupled device (CCD) cameras convert photons into digital signals using a light-sensitive silicon chip. Many times, multiple exposures (2-4) must be done to obtain an image in the linear range, increasing the cost of the experiment. In addition to the costs of maintaining the dark room and purchasing chemicals, film can cost up to $2/sheet. Although the film can be scanned and densitometry used to quantify signals relative to a loading control, this is generally not regarded as a quantitative measurement due to the limitations of film.Īlthough many laboratories have access to a communal developer and do not have to purchase their own, using film for Western blots is expensive. Film has a narrow linear range and is easily saturated by moderate to high signal intensities. However, a major drawback to film is that it is only semi-quantitative. The film can then be developed within minutes using a commercial developer.įilm is ideal for documenting relative amounts of proteins, such as assessing whether a protein is expressed by a particular tissue or cell line, or when comparing large signal differences. The exposure time can be adjusted easily to allow for short exposures and capture of high intensity signals or long exposures for low intensity signals. The close apposition of the blot to the film allows for sensitive detection of the signal. When used with ECL, the x-ray film is placed directly against the plastic-wrapped blot. For example, Advansta’s LucentBlue x-ray film is economically priced for use in chemiluminescent Western blotting. In addition, companies have developed films with enhanced sensitivity to the light emitted during the chemiluminescent reaction. The light emitted during development with ECL substrates is easily captured by x-ray film. However, when exposed to a chemical developing solution during the developing process, the reaction forms black, metallic silver that is suspended in the gelatin emulsion and creates the image. Normally this change is so small it remains undetected. When x-rays or light hit the crystals, some of the Br- or Cl- ions are freed and captured by the Ag+ ions. X-ray film is composed of a flexible, transparent base coated with a gelatin emulsion containing radiation-sensitive halide crystals (usually silver bromide or silver chloride). This Advansta WiKi Guide will discuss the advantages and disadvantages of film and digital imaging. However, digital imaging has given researchers new methods for documenting and analyzing Western blots. Traditionally, the light was captured and documented on x-rays films. ECL relies on the production of light through an enzyme–substrate interaction. Since the late 1970’s, ECL has replaced radioactive probes and less-sensitive colorimetric assays. Chemiluminescent Western blotting (also called enhanced chemiluminescence ECL) is one of the most popular methods for detecting protein expression.
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