![]() This underlines the negative perception by which the scientific community views the western blot data. A recent report predicts that approximately 25 % of the accepted papers include at least one inappropriately manipulated figure and many of these are associated with western blotting. These include challenges related to every step of the western blotting procedure, from sample preparation, normalization, SDS–PAGE gel loading, protein transfer, primary and secondary antibody selection, incubations, and washes, detection method selection to densitometric analysis. However, there are many potential stumbling blocks in this procedure that can preclude reliable results. This multistep method determines the presence or absence, size, and modification or degradation states of target proteins, as well enables the quantitation of proteins from complex protein mixtures or homogenates. J Med Microbiol 46: 314-320.Western blotting has been a staple in life science labs for several decades-ever since researchers published the first detailed description of this protein detection technique in 1979. Direct comparison of two commercially available computer programs for analysing DNA fingerprinting gels. Seward RJ, Ehrenstein B, Grundmann HJ, Towner KJ (1997). Two-dimensional gel electrophoretic analysis of vectorially labeled surface proteins of human spermatozoa. Naaby-Hansen S, Flicklinger C, Herr JC (1997). Image-Pro ®®Plus Version 1.0 for Windows ™, Reference Manual, Media Cybernetics Inc. Reproducibility of digital image analysis for measuring corneal haze after myopic photorefractive keratectomy. Maldondo M, Arnau V, Martinez-Costa R, Navea A, Mico F, Cisneros A, Menezo J (1997). A muscle hypertrophy condition in lamb (callipyge): Characterization of effects on muscle growth and meat quality traits. Koohmaraie M, Shackelford SD, Wheeler TL, Lonergan SM, Doumit ME (1995). A simplified method of analysis of cell-conditioned medium for Insulin-like Growth Factor-I (IGF-I) activity. Krabbenhoft EA, O'Reilly BA, Shultz K, Chen Y, Stewart NT, Dodson MV (1997). In: Protein Functionality in Food Systems, New York: Marcel Dekker Inc, pp 79-119. Protein separation and analysis of certain skeletal muscle proteins: Principles and Techniques. Huff-Lonergan EJ, Beekman DD, Parrish Jr FC (1994). Evaluation of satellite cell cultures by computer/video imaging enhancement: An undergraduate research project. Howard JH, Vierck J, Howell S, Dodson MV (1993). Wound status evaluation using color image processing. Hansen G, Sparrow E, Kokate J, Leland K, Iaizzo P (1997). Protein Blotting: A guide to transfer and detection. Collectively, the results of these two studies suggest that specific proteins may be evaluated by using relatively inexpensive image analysis software systems via pixel quantification of electronic images.īio-Rad Laboratories (1996). A second image analysis program, Alpha Imager™ 2000, was then used to define the boundaries of protein bands, assess pixel number and density, and to obtain final numerical data for quantifying α-Actinin on the WB. In the second procedure, WB were scanned with a ScanJet 3c® flat bed scanner and their backgrounds were clarified using Image-Pro® Plus. Dot blots corresponding to a linear concentration range from 10 to 300 ng IGF-Iwere assessed by this method. After the DB were developed and dried, the images were digitized using an HP Deskscan II® flat bed scanner, exported into Image-Pro® Plus and analyzed by taking the combined mean of 45° and 135° sample lines drawn through each dot. In the first procedure, known IGF-I samples were dotted on nitrocellulose membranes using a vacuum manifold. Inexpensive computer imaging technology was used to assess levels of insulin-like growth factor-I (IGF-I) on dot blots (DB) and α-Actinin on Western blots (WB). ![]()
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