Analytical ultracentrifugation with fluorescence detection and biosafety containment and its application to the prion protein

  • M. Pitschke
  • K. Post
  • D. Riesner
Biological Systems
Part of the Progress in Colloid & Polymer Science book series (PROGCOLLOID, volume 107)


In order to utilize the high sensitivity of the fluorescence detection system for sedimentation equilibrium in analytical ultracentrifugation, a new evaluation method was developed. In comparison to absorption recording the sensitivity could be raised 100–1000 fold as shown for a series of proteins (bovine serum albumin, immunoglobulin G and chymotrypsin). Sedimentation equilibrium runs can be analysed down to sample concentrations of 0.25 ng/µl with a minimal sample volume of 40 µl. The accuracy of the molecular weights is comparable to that recorded by the absorption optics. Analysis of infectious prions in the analytical ultracentrifuge requires strict safety conditions. A biohazard safety containment with a vacuum system is constructed to prevent contamination of the laboratory. Decontamination of the cells is ensured by the use of self-manufactured titanium cells, which could be autoclaved at 134 °C in 1 N NaOH. Analysis of SDS-solubilized PrP(27–30) which is a N-terminal truncated fragment of the whole prion protein, showed by anlytical ultracentrifugation relatively homogeneous fractions with molecular masses between 20.000 Da and 120.000 Da, depending on the conditions of solubilization.

Key words

Molecular weight determination analytical ultracentrifugation fluorescence detection prion protein fluorescence labelling 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Rappold W (1986) Ph D thesis, Universität DüsseldorfGoogle Scholar
  2. 2.
    Schmidt B, Rappold W, Rosenbaum V, Fischer R, Riesner D (1990) Colloid Polym Sci 268:45–54CrossRefGoogle Scholar
  3. 3.
    Gajdusek DC (1977) Science 197:943–960PubMedCrossRefGoogle Scholar
  4. 4.
    Parry HB (1983) In: Oppenheimer DR (ed) Academic Press, New York, pp. 1–192Google Scholar
  5. 5.
    Pruisiner SB (1982) Science 216:136–144CrossRefGoogle Scholar
  6. 6.
    Wells GAH, Wilesmith (1995) Brain Pathol. 5:91–103PubMedCrossRefGoogle Scholar
  7. 7.
    Kellings K, Meyer N, Mierenda C, Prusiner SB, Riesner (1992) J Gen Virol 73:1025–1029PubMedCrossRefGoogle Scholar
  8. 8.
    Prusiner SB (1991) Science 252:1515–1522PubMedCrossRefGoogle Scholar
  9. 9.
    Prusiner SB, McKinley MP, Bowman KA, Bolton DC, Bendheim PE, Groth DF, Glenner GG (1983) Cell 35:349–358PubMedCrossRefGoogle Scholar
  10. 10.
    Riesner D, Kellings K, Post K, Wille H, Serban H, Groth D, Baldwin MA, Prusiner SB (1996) J. Virol 70:1714–1722PubMedGoogle Scholar
  11. 11.
    Floßdorf J (1980) Makromol Chem 181:715–724CrossRefGoogle Scholar
  12. 12.
    Yphantis DA (1964) Biochemistry 3:297PubMedCrossRefGoogle Scholar
  13. 13.
    Durchschlag H (1986) In Hinz, HJ (ed). Springer, Berlin, pp. 45–128Google Scholar
  14. 14.
    Durchschlag H (1989) Colloid Polym Sci 267:1139–1150CrossRefGoogle Scholar

Copyright information

© Dr. Dietrich Steinkopff Verlag GmbH & Co. KG 1997

Authors and Affiliations

  • M. Pitschke
    • 1
  • K. Post
    • 1
  • D. Riesner
    • 1
  1. 1.Institute für Physikalische BiologieHeinrich-Heine-Universität DüsseldorfDüsseldorfGermany

Personalised recommendations