Probing with a loading control can be carried out alongside the target antibody by cutting the membrane before antibody incubation, or after the blot has been stripped of previously bound antibodies. Perform titration experiments on your samples to determine the concentration of antibody against your housekeeping protein and blot exposure time that gives optimum signal-to-background ratio without signal saturation. Housekeeping proteins are often highly expressed, making it easy for the signal from the housekeeping protein detection to become saturated and affect normalization. If you are performing a multiplexed fluorescent western blot experiment, choose your brightest fluorophore for your protein of interest and your dimmest fluorophore for your housekeeping protein. This will prevent overexposure and therefore oversaturation of the signal from your housekeeping protein.īeta Actin is a 42 kDa protein ubiquitously expressed in all eukaryotic cells. J Med Microbiol 46: 314-320.It plays a critical role in the regulation of cell migration and gene expression through control of the cellular G-actin pool (Bunnell et al. 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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