Abstract
This report discusses recent methods of sample preparation and gel electrophoresis for 35S immunoprecipitation (IP) and IP western blotting. In both methods, IP is used to obtain purified proteins, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate the proteins on a gel. In 35S IP, the proteins are radiolabeled and visualized on film by fluorography; in IP blotting, proteins are transferred onto nitrocellulose paper, and antibodies are used to detect specific proteins. A similar IP and SDS-PAGE method can be used for both procedures, but IP blotting has the potential advantages of improvement in sensitivity for low-abundance proteins and enhanced specificity for identification of proteins from a mixture. Some of the technical adaptations discussed here to facilitate IP blotting and avoid loss of beads or purified proteins may also be useful for 35S IP.
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References
Ornstein L (1964) Disc electrophoresis. I. Background and theory. Ann N Y Acad Sci 121:321–349
Kohlrausch F (1897) Ueber concentrations-verschiebungen durch electrolyse im inneren von lösungen und lösungsgemischen. Ann Phys Chem 62:209–239
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Pollard JW (1994) The in vivo isotopic labeling of proteins for polyacrylamide gel electrophoresis. Methods Mol Biol 32:67–72
Paulsson KM, Jevon M, Wang JW et al (2006) The double lysine motif of tapasin is a retrieval signal for retention of unstable MHC class I molecules in the endoplasmic reticulum. J Immunol 176:7482–7488
Targoff IN, Mamyrova G, Trieu EP et al (2006) A novel autoantibody to a 155-kd protein is associated with dermatomyositis. Arthritis Rheum 54:3682–3689
Trieu EP, Gross JK, Targoff IN (2009) Immunoprecipitation-western blot for protein of low abundance. In: Kurien BT, Hal Scofield R (eds) Protein blotting and detection: methods and protocols, vol 536, Methods in molecular biology., pp 259–275
Hames BD (ed) (1998) Gel electrophoresis of proteins: a practical approach, 3rd edn, The practical approach series. Oxford University Press, Oxford
Horscroft NJ, Roy P (1997) Thermal denaturation of proteins for SDS-PAGE analysis by microwave irradiation. Biotechniques 22: 224–226
Bonner WM (1987) Autoradiograms: 35S and 32P. Methods Enzymol 152:55–61
Laskey RA (1994) Radiometric methods for detection in blots. In: Dunbar BS (ed) Protein blotting: a practical approach, The practical approach series (Rickwood D and Hames BD, eds). Oxford University Press, Oxford, pp 121–132
Kessler SW (1981) Use of protein A-bearing staphylococci for the immunoprecipitation and isolation of antigens from cells. Methods Enzymol 73:442–459
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Trieu, E.P., Targoff, I.N. (2012). SDS-PAGE for 35S Immunoprecipitation and Immunoprecipitation Western Blotting. In: Kurien, B., Scofield, R. (eds) Protein Electrophoresis. Methods in Molecular Biology, vol 869. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-821-4_18
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DOI: https://doi.org/10.1007/978-1-61779-821-4_18
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