Major challenges for the reliable protein quantification by Western blotting are adequate data normalization and stable protein detection. Nevertheless, with the development of fast blotting systems and further development of immunostaining methods, a reduction of the processing time can be achieved. However, the whole procedure is often very time-consuming. It enables detection of a target protein based on the use of specific antibodies. 237, 123–128.For the quantification of certain proteins of interest within a complex sample, Western blot analysis is the most widely used method. (1996) Comparison of reflectance and transmission densito-metry, using document and laser scanners, for quantitation of stained western blots. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some appliactions, Proc. Towbin, H., Staehelin, T., and Gordon, J. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. (1990) The use of charge coupled devices in the quantitative evaluation of images, on photographic film or membranes, obtained following electrophoretic separation of DNA fragments. (1980) The use of intensifying screens or organic scintillators for visualizing radioactive molecules resolved by gel electrophoresis. (1992) Quantitative dot-blot assay for proteins using enhanced chemiluminescence. (1998) Characterization of acrid-ancarboxylic acid derivatives as chemiluminescent peroxidase substrates. A., Sugioka, K., Sugioka, Y., and Schaap, A. Nature 346, 297.Īkhavan-Tafti, H., DeSilva, R., Arghavani, Z., Eickholt, R. (1990) Light-based detection of biomolecules. (1994) Molecular Dynamics Application Note #57, Molecular Dynamics Inc.ĭurrant, I. (2000) Multiplex detection and quantitation of proteins on Western blots using fluorescent probes. (1998) Quantitative measurement of proteins by Western blotting with Cy5-coupled secondary antibodies. (1991) Chemiluminescence detection of proteins from single cells. (1997) Determination of aryl hydrocarbon receptor nuclear translocator protein concentration and subcellular localization in hepatic and nonhepatic cell culture lines: Development of quantitative Western blotting protocols for calculation of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein in total cell lysates. (1985) Protein and nucleic acid blotting and immunobiochemical detection. This process is experimental and the keywords may be updated as the learning algorithm improves.īers, G. These keywords were added by machine and not by the authors. However, it is also possible to use Western blotting to provide accurate quantification of proteins in samples, to assess changes in protein expression levels ( 2). Information can be obtained about the molecular weight of the protein of interest and its relative abundance in the sample. The major application of Western blotting has been the detection of individual proteins in complex mixtures such as cell lysates. The label may be radioactive, colorimetric, fluorescent, or chemiluminescent. A labeled secondary antibody is then added, which binds to the primary antibody. Western blots are detected with antibodies specific to the target protein which are known as primary antibodies. The technique of Western blotting, the electrophoretic transfer of proteins from sodium dodecyl sulfate (SDS)-polyacrylamide gels to membranes, is a core technique in molecular and cell biology, see ref.

Quantify western blot