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Western Blot Membrane Detection Methods. Information on the various protocols used to analyze and expose films or come up with data from membranes. This includes: chemiluminescence detection, ECL, colorimetric, radioactive, and fluorescent detection of blots.
Western Blot Membrane Detection Methods
A western blot also called immunoblotting is a molecular biology technique used to detect a specific protein in a given cell culture or tissue extract sample. Proteins are first separated by molecular weight on an SDS-PAGE by gel electrophoresis and then transferred to a membrane. See Western Blot Transfer and Western Blot gel electrophoresis. Once the proteins in the gel are transferred to a membrane, the membrane is incubated or "probed" with primary and secondary antibodies. The primary antibody is specific to the protein of interest, whereas the secondary antibody usually contains an enzyme or factor which allows for various methods of detection. Most commonly the secondary antibody is a modified antibody which is linked to a reported enzyme (usually HRP - or horse radish peroxidase) which is exposed to substrate which in the presence of the enzyme produces localized light in the region where the antibody is bound to the membrane (i.e the spot on the membrane where the protein of interest is!). Traditionally western blot detection takes place with a two-step process, however there are now some one-step detection methods available. Methods of Detection in a Western Blot include: Chemiluminescence or ECL Detection - Western Blots By far the most common method, but which may be replace chemiluminescent detection for western blots require incubation of the membrane a substrate that will luminesce when exposed to the enzyme present on the (modified) secondary antibody. The localized light which is emitted from the bands is detected by photosensitive photographic film. Developments recently have allowed CCD camera capturing of the light emission from the membrane and a more quantitative analysis of the membrane as a digital image of the exposed western blot membrane. Images are then analysed by densitometry. Densitometry allows the gathering of data from the density of the spots on the film and analyzes the quantifies the results in terms of optical density or OD or O.D in densitometry.
Relatively new densitometry software further allows membrane data analysis with molecular weight analysis if standards are present on the membrane. "Enhanced ChemiLuminescent" also called ECL detection is now considered to be among the most sensitive detection methods for western blotting. Colorimetric Detection - Western Blots Colorimetric detection protocols for western blot membranes depend on the incubation of the membrane with a substrate that reacts with the reporter enzyme present on the secondary antibody. The enzyme then catalyzes the conversion of the substrate into an insoluble coloured dye that precipitates in a localized area in the vicinity of the bound antibody. The reaction is stopped by washing or removing the substrate from the enzyme. The colour stains the nitrocellulose membrane and can its intensity can be detected by densitometry methods similar to ECL or spectrophotometry. Radioactive detection - Western Blots One of the most quantitative methods however not used much is radioactive detection of western blot membranes. Instead of enzyme-linked antibodies, the secondary or even primary antibodies contain a radioactive label. This radioactive label can be 32-P, I-125 or similar radioactivity emitting element. The radioactive emissions are then detected by medical or X-ray sensitive film which is placed against the western blot membrane. The film is developed in the dark, similarly to chemiluminescent western blot detection. The film is exposed to the membrane, and the localized radioactivity emitted from the antibodies bound to the proteins exposed the film in the region to create a dark band. Advantages of Radioactive Detection of Western Blots - sensitive
- a gold standard for quantitative analysis
Disadvantages of Radioactive Detection of Western Blots Due to the fact that radioactivity is fairly costs, and a health / safety risk, its usage is in decline in western blot detection (and also other methods) in favour of non-radioactive detection methods which are proving to be as (or even more) sensitive to radioactive detection methods for western blots. Fluorescent detection - Western Blots Fluorescnet methods of detection seem to be the future of western blotting. In western blot detection, fluorescent labeled probes are excited by light. A CCD camera equipped with a photosensor and emission filters captures the emission of the excitation. Subsequently, a digital image of the membrane is captured and the western blot spot emission data is analyzed by molecular weight, and emission data detection allows for quantitative analysis. Fluorescent detection methods are considered to be among the most sensitive and qualitative detection methods for western blot membrane analysis. Secondary probing - Western Blots An important difference between PVDF and nitrocellulose membranes is the ability of the membranes to support stripping of the antibodies off the membrane and reprobing (reuse of the membrane for subsequent western blotting). An advantage of PVDF is it allow for easier stripping and can be used more times than nitrocellulose befeore increasing background noise limits subsequent detection.
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