Table 1.

Summary of in vitro assays for PrP^C conversion and PrP-res formation.

Conversion Method	Conversion Buffer	Incubation	Sonication/ Agitation	PrPC Source	PrPSc Source	Percent Converted/ Amplified	Infectivity	Reference
Mixing	PBS with protease inhibitors	37°C ≤ 24 hr		Lysate of N2a cells expressing MHM2 PrPC	PrP27-30 purified from prion-infected mouse brains	0%		7
Metabolic Radiolabeling	PBS with protease inhibitors	37°C ≤ 24 hr		Lysate of ScN2a cells expressing [35S]-PrPC	Endogenous PrPSc of ScN2a cells	0%		7
Microsomal Membranes	20 mM Tris buffer, pH 7.5	25°C 1 hr		[35S]-PrPC synthesized by cell-free translation systems	Microsomal membranes from scrapie-infected hamster brain cells.	0%		7
Cell-Free Conversion	0.75 M GdnHCl, 130 mM NaCl, 10 mM Tris-HCl, pH 7.0	20°C 22 hr		[35S]-PrPC expressed in mouse fibroblast cells	Brain-derived PrPSc treated with 2 -3 M GdnHCl for 5 h at 37°C	10-20% of PrPC converted	No	8
Cell-Lysate Conversion	50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 0.5%SDS	37˚C 48 hr		Lysate of CHO cells expressing MHM2 PrPC	Brain-derived mouse PrPSc	Successful, not quantified		24
PMCA	PBS with 0.05% Triton X-100, 0.05% SDS, protease inhibitors	37˚C 10-72 hr	40 sec sonication	Normal, uninfected crude brain homogenate	Prion-infected crude brain homogenate	~ 20 -100 fold increase of PrPSc	Yes	26, 27
PMCA under non-denaturing conditions	PBS with 1% Triton-X 100, 0.5 mM EDTA	37˚C 16-48 hr	Continuous agitation, 800 rpm	Purified brain -derived PrPC	PrP27-30	~10-fold increase of PrPSc	Yes	29, 30
rPrP-PMCA	PBS with 0.05-0.1% SDS, 0.05-0.1% Triton X-100	37˚C 24 hr	40 sec sonication	rPrPC expressed in E.coli	Purified PrPSc or crude homogenate of prion-infected brains	~10% of rPrPC converted; fold increase of PrPSc not quantified		34
QUIC	PBS with 0.05% SDS, 0.05% Triton X-100	45˚C 46 hr	10 sec agitation, every 2 min	rPrPC expressed in E.coli	Prion-infected crude brain homogenate	Variable, sensitive to environmental conditions		35
Autocatalytic Conversion Assay	β-oligomer : sequential dilution with 5 M urea, 20 mM NaOAc‡, 0.2 M NaCl, pH 3.7, and with 1 M urea, 20 mM NaOAc, 0.2 M NaCl, pH 5.5	20˚C 16 hr		rPrPC expressed in E.coli	None			36
	amyloid fibril : identical buffer to generate β-oligomer†	37˚C 10-72 hr	Continuous agitation, 600 - 900 rpm	rPrPC expressed in E.coli	None		Yes	38, 40

^† The buffer system has been improved for the recent studies in which buffer containing 1 M GdnHCl, 2.4 M urea, and 150 mM NaCl, pH5.0-6.8 was used ³⁷-³⁹. ^‡ NaOAc: sodium acetate.