Imaging and data analysis is an essential step in the western blotting workflow. Western blots can be imaged and analyzed using different methods. This page reviews some of these methods such as luminescence detection, fluorescence imaging, densitometery, and autoradiography. It also reviews some of the analysis software that Bio-Rad offers and their features.
Related Topics: Protein Blotting Methods, Protein Blotting Equipment, Membranes and Blotting Papers, Transfer Buffers, and Transfer Conditions.
Several instruments and devices are employed to document western blotting results (see table below).
Comparison of western blot documentation and analysis methods.
For chemiluminescence detection, CCD imaging is the easiest, most accurate, and rapid method. Traditionally, the chemiluminescent signal from blots was detected by X-ray film. Film is a sensitive medium for capturing the chemiluminescent signal but suffers from a sigmoidal response to light with a narrow region of linear response, which limits its dynamic range. To gather information from a blot that has both intense and weak signals, multiple exposures are required to produce data for all samples in the linear range of the film. A process termed preflashing can improve linearity but this requires extra equipment and effort. Additionally, quantitation of data collected by exposure to film requires digitization (that is, scanning of X-ray film with a densitometer).
CCD cameras have a linear response over a broad dynamic range – 2-5 orders of magnitude – depending on the bit depth of the system. CCD cameras also offer convenience by providing a digital record of experiments for data analysis, sharing, and archiving, and by eliminating the need to continually purchase consumables for film development. Some CCD imagers offer chemiluminescent sensitivity equal to film.
Fluorescence, chemifluorescence, and colorimetric detection all benefit from the advantages of digital imaging: convenience, digital records of experiments, sensitive, and wide dynamic ranges. Fluorescent and chemifluorescent signals can be detected with different types of imaging systems, including CCD and laser-based technologies. For example, the ChemiDoc™ MP and PharosFX™ Plus systems can be used similarly to detect fluorescent and chemifluorescent signals. The decision to use one type of technology over another depends on budget and requirements for limit of detection and resolution. CCD systems are generally less expensive than laser-based systems. The resolution of CCD and laser-based systems can be similar, with the finest resolution settings of 50 µm when used for gels and blots. Another advantage of fluorescence and chemifluorescence detection is that the detection limits and dynamic range of CCD and laser-based systems generally far exceed the dynamic ranges of the fluorescence assays currently used for protein detection.
Colorimetric samples can be easily recorded and analyzed with a densitometer such as the GS-800™ calibrated densitometer. The densitometer provides a highly reproducible digital record of the blot with excellent image resolution, and accurate quantitation. The GS-800 densitometer uses red, green, and blue color CCD technology to enhance detection of a wide range of colorimetric staining reagents.
To detect the commonly used beta emitting radioisotopes, 35S, 32P, 33P, 12C, and 125I, the most widely used method is autoradiography on X-ray film. Autoradiography provides a good combination of sensitivity and resolution without a large investment in detection substrates or imaging systems. For direct autoradiography the response of the film is linear only within a range of 1-2 orders of magnitude. Use of intensifying screens and fluorographic scintillators can increase sensitivity, and pre-exposing film to a flash of light can improve linearity. However, these measures to improve signal detection are limited. Phosphor imagers, such as Bio-Rad's PharosFX Plus system or Personal Molecular Imager™ (PMI) system, offer an alternative in detecting gels and blots labeled with beta emitting radioisotopes. The initial investment in instrumentation frees up time and resources associated with using X-ray film and offers increased sensitivity, wider dynamic range and 10 to 20 times shorter exposure times than those for X-ray film detection. The ability to accurately quantitate data is much greater with storage phosphor screens because the linear dynamic range of phosphor imagers is significantly greater – 5 orders of magnitude – enabling accurate quantitation and the elimination of over-exposure and saturated signals.
Blot detection using an imaging system needs a robust software package for image acquisition. In addition, a good software package can magnify, rotate, resize, overlay, and annotate the corresponding gel and blot images, allowing export of the images to common documentation software. A good software package also allows analysis of the blot image and comparisons of relative signal intensities, protein molecular weight, or other aspects.
For automated acquisition and analysis of gel and blot images, Bio-Rad offers:
Image Lab software. E. coli lysate was separated and activated on a 4–20% Criterion™ TGX Stain-Free™ gel and transferred onto a PVDF membrane. The membrane was imaged on a Gel Doc™ EZ system and analyzed using Image Lab 3.0 software.
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