Abstract
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.
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Rappold W (1986) Ph D thesis, Universität Düsseldorf
Schmidt B, Rappold W, Rosenbaum V, Fischer R, Riesner D (1990) Colloid Polym Sci 268:45–54
Gajdusek DC (1977) Science 197:943–960
Parry HB (1983) In: Oppenheimer DR (ed) Academic Press, New York, pp. 1–192
Pruisiner SB (1982) Science 216:136–144
Wells GAH, Wilesmith (1995) Brain Pathol. 5:91–103
Kellings K, Meyer N, Mierenda C, Prusiner SB, Riesner (1992) J Gen Virol 73:1025–1029
Prusiner SB (1991) Science 252:1515–1522
Prusiner SB, McKinley MP, Bowman KA, Bolton DC, Bendheim PE, Groth DF, Glenner GG (1983) Cell 35:349–358
Riesner D, Kellings K, Post K, Wille H, Serban H, Groth D, Baldwin MA, Prusiner SB (1996) J. Virol 70:1714–1722
Floßdorf J (1980) Makromol Chem 181:715–724
Yphantis DA (1964) Biochemistry 3:297
Durchschlag H (1986) In Hinz, HJ (ed). Springer, Berlin, pp. 45–128
Durchschlag H (1989) Colloid Polym Sci 267:1139–1150
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© 1997 Dr. Dietrich Steinkopff Verlag GmbH & Co. KG
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Pitschke, M., Post, K., Riesner, D. (1997). Analytical ultracentrifugation with fluorescence detection and biosafety containment and its application to the prion protein. In: Jaenicke, R., Durchschlag, H. (eds) Analytical Ultracentrifugation IV. Progress in Colloid & Polymer Science, vol 107. Steinkopff. https://doi.org/10.1007/BFb0118016
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DOI: https://doi.org/10.1007/BFb0118016
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