Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Burnette WN (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.Burnette in 1981 after the eponymous Southern blot for DNA and the consequent coinage of the northern blot in 1977 for RNA.12 Western blotting separates, detects, and identifies one or more proteins in a complex mixture. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. The name ‘western’ blot was first coined by Dr.YALOW RS, GLICK SM, ROTH J, BERSON SA (1964) Radioimmunoassay of human plasma ACTH.This tool will allow the user to adjust the contrast and brightness of the image. The use of film for western blot detection is slowly disappearing because of non linearity of the image (non accurate quantification). Once the image is open, the user will need to select the Levels tool from the Image menu. The light is then detected by CCD cameras which capture a digital image of the western blot or photographic film. Sarge KD, Park-Sarge O-K (2009) Detection of proteins sumoylated in vivo and in vitro. In order to edit a western blot image, the first step is to open the image in an image editing program such as Adobe Photoshop.Biochim Biophys Acta, Mol Cell Res 1833:812–822 Voelkel T, Andresen C, Unger A et al (2013) Lysine methyltransferase Smyd2 regulates Hsp90-mediated protection of the sarcomeric titin springs and cardiac function.Pere-Brissaud A, Blanchet X, Delourme D et al (2015) Expression of SERPINA3s in cattle: focus on bovSERPINA3-7 reveals specific involvement in skeletal muscle. Electrophoretic gels such as Western blots need frequently to be quantified in order to translate biochemical results into statistical values (see Gels ).Conte A, Sigismund S (2017) Methods to investigate EGFR ubiquitination.Wu WC, Walaas SI, Nairn AC, Greengard P (1982) Calcium/phospholipid regulates phosphorylation of a Mr “87k” substrate protein in brain synaptosomes.The proposed method represents an affordable, accurate and reproducible approximation that could be used in the presence of limited resources availability.īackground subtraction Densitometry ImageJ Quantification Western blot.Ĭopyright © 2018 Elsevier B.V. Each pixel in a blot image has an x and y coordinate, in addition to an. 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. This is what you get when you treat each row in the dot blot as a horizontal 'lane' and use the gel analysis procedure in the ImageJ manual. A protein band is a feature that appears in a western blot image. In this work, we describe the use of office scanner coupled with the ImageJ software together with a new image background subtraction method for accurate Western blot quantification. This dot blot image is available in the File/Open Samples menu in ImageJ 1.33s or later. Furthermore, the use of semi-quantitative software as ImageJ (Java-based image-processing and analysis software) is clearly increasing in different scientific fields. Due to the technical knowledge required for densitometry analysis together with the resources availability, standard office scanners are often used for the imaging acquisition of developed Western blot films. Western blot quantification method constitutes a critical step in order to obtain accurate and reproducible results. It constitutes a multistep method that allows the detection and/or quantification of proteins from simple to complex protein mixtures. Since its first description, Western blot has been widely used in molecular labs. This protocol offers an Image J macro/plugin that enable easy quantification of bands on western blots, dot blots, and fluorescent gels etc.
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