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Nonradioactive Methods for Visualization of Protein Blots

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Book cover Immunochemical Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 80))

Abstract

The ability to detect specific proteins that have been immobilized on nitrocellulose or polyvinylidene difluoride (PVDF) membranes has become an invaluable technique in all areas of the biological sciences. In the past, visualization of low-abundance proteins required the use of radioactive markers, such as 125Iodine, but with the advent of enhanced chemiluminescent technology and amplified colorimetric detection, this is no longer the case (13). This chapter details two of the more popular nonradioactive methods for immobilized protein detection, both of which rely on the indirect labeling of specific proteins with enzyme-linked antibodies. The location and abundance of these enzyme markers are then realized in a reaction producing a colored end product or by the generation of light in a chemiluminescent reaction. These techniques can identify <20 pg of protein.

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References

  1. Whitehead, T. P, Thorpe, G. H G, Carter, T. J. N, Groucutt, C, and Kricka, L J. (1983) Enhanced luminescence procedure for sensitive determination of peroxidase-labeled conjugates in immunoassay. Nature 305, 158,159.

    Article  CAS  Google Scholar 

  2. Thorpe, G. H G. and Kricka, L J (1986) Enhanced chemiluminescent reactions catalysed by horseradish peroxidase. Methods Enzymol 133, 331–353.

    Article  PubMed  CAS  Google Scholar 

  3. Pluzek, K.-J. and Ramlau, J (1988) Alkaline phosphatase labeled reagents, in CRC Handbook of Immunoblotting of Proteins, vol. 1, Technical Descriptions (Bjerrum, O.J and Heegaard N H H., eds.), CRC, Boca Ration, FL, pp. 177–188

    Google Scholar 

  4. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

    Article  PubMed  CAS  Google Scholar 

  5. Hames, B. D. and Rickwood, D (eds) (1990) Gel Electrophoresis of Proteins, 2nd ed, IRL, Oxford, UK

    Google Scholar 

  6. Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylarmde gels to nitrocellulose sheets’ procedure and some applications. Proc Natl Acad. Sci USA 76, 4350–4354.

    Article  PubMed  CAS  Google Scholar 

  7. Kyhse-Anderson, J. (1984) Electroblotting of multiple gels—a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J. Biochem Biophys. Methods 10, 203–209.

    Article  Google Scholar 

  8. Lauriere, M (1993) A semidry electroblotting system efficiently transfers both high-molecular-weight and low-molecular-weight proteins separated by SDS-PAGE. Anal Biochem 212, 206–211

    Article  PubMed  CAS  Google Scholar 

  9. “ECL™ western blotting protocols” Amersham International plc. Bucks, UK.

    Google Scholar 

  10. Millipore Technical Protocols 006/007 (1987) Millipore Corporation, Bedford, MA

    Google Scholar 

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© 1998 Humana Press Inc., Totowa, NJ

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Finney, M. (1998). Nonradioactive Methods for Visualization of Protein Blots. In: Pound, J.D. (eds) Immunochemical Protocols. Methods in Molecular Biology™, vol 80. Humana Press. https://doi.org/10.1007/978-1-59259-257-9_20

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  • DOI: https://doi.org/10.1007/978-1-59259-257-9_20

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-493-8

  • Online ISBN: 978-1-59259-257-9

  • eBook Packages: Springer Protocols

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