Springer Protocols: Abstract: 3. Immunodetection of PrPSc Using Western and Slot Blotting Techniques

3. Immunodetection of PrPSc Using Western and Slot Blotting Techniques

By: Hanna Gyllberg³, Kajsa Löfgren³

Abstract

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Prion infectivity is often linked to presence of the protease-resistant isoform of prion protein (PrP), PrP^res; therefore, it is of highest interest to have convenient methods for rapid detection of PrP^res in the research laboratory. For detection of PrP^res in model systems to confirm infectivity, there are several methods that can be applied. This chapter focuses on detection of PrP^res by proteinase K digestion followed by Western blot, which is the only method that is both quantitative and qualitative. For large-scale screening of PrP^res content in samples, the dot blot method offers a great advantage for detecting PrP^res, and this method is also thoroughly described in this chapter.

Affiliation(s): (3) Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

Book Title: Prion Protein Protocols

Series: Methods in Molecular Biology | Volume: 459 | Pub. Date: Jun-04-2008 | Page Range: 35-48 | DOI: 10.1007/978-1-59745-234-2_3

Subject: Protein Science

Key Words: Dot blot - guanidinium thiocyanate - immunoprecipitation - nitrocellulose membrane - proteinase K digestion - PrP antibodies - PVDF membrane - reprobing - Western blot

References

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1.	Pan, K. M., Baldwin, M., Nguyen, J., Gasset, M., Serban, A., Groth, D., Mehlhorn, I., Huang, Z., Fletterick, R. J., Cohen, F. E., et al. (1993). Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci U S A 90, 10962–10966.

2.	Prusiner, S. B. (1998). Prions. Proc Natl Acad Sci U S A 95, 13363–13383.

3.	McKinley, M. P., Meyer, R. K., Kenaga, L., Rahbar, F., Cotter, R., Serban, A., and Prusiner, S. B. (1991). Scrapie prion rod formation in vitro requires both detergent extraction and limited proteolysis. J Virol 65, 1340–1351.

4.	Weissmann, C. (2004). The state of the prion. Nat Rev Microbiol 2, 861–871.

5.	Oesch, B., Jensen, M., Nilsson, P., and Fogh, J. (1994). Properties of the scrapie prion protein: quantitative analysis of protease resistance. Biochemistry 33, 5926–5931.

6.	Aguzzi, A., Heikenwalder, M., and Miele, G. (2004). Progress and problems in the biology, diagnostics, and therapeutics of prion diseases. J Clin Invest 114, 153–160.

7.	Collinge, J., Sidle, K. C., Meads, J., Ironside, J., and Hill, A. F. (1996). Molecular analysis of prion strain variation and the aetiology of ‘new variant’ CJD. Nature 383, 685–290.

8.	Blochberger, T. C., Cooper, C., Peretz, D., Tatzelt, J., Griffith, O. H., Baldwin, M. A., and Prusiner, S. B. (1997). Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system. Protein Eng 10, 1465–1473.

9.	Cancellotti, E., Wiseman, F., Tuzi, N. L., Baybutt, H., Monaghan, P., Aitchison, L., Simpson, J., and Manson, J. C. (2005). Altered glycosylated PrP proteins can have different neuronal trafficking in brain but do not acquire scrapie-like properties. J Biol Chem 280, 42909–42918.

10.	Serban, D., Taraboulos, A., DeArmond, S. J., and Prusiner, S. B. (1990). Rapid detection of Creutzfeldt-Jakob disease and scrapie prion proteins. Neurology 40, 110–117.

11.	Gasset, M., Baldwin, M. A., Fletterick, R. J., and Prusiner, S. B. (1993). Perturbation of the secondary structure of the scrapie prion protein under conditions that alter infectivity. Proc Natl Acad Sci U S A 90, 1–5.

12.	Beringue, V. , Vilette, D., Mallinson, G., Archer, F., Kaisar, M., Tayebi, M., Jackson, G. S., Clarke, A. R., Laude, H., Collinge, J., et al. (2004). PrPSc binding antibodies are potent inhibitors of prion replication in cell lines. J Biol Chem 279, 39671–39676.

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