Figure 10.

Return refocusing gel electrophoresis. (A) The first electrophoresis is a conventional PAGE to separate macromolecules of a prion sample. Nucleic acids (black bars) and proteins (hollow bars) migrate according to their charge and size. After electrophoresis the gel is cut into slices marked a–e. (B) The slices are polymerized at the bottom of a new gel matrix containing 1% SDS. (C) In a second electrophoresis in the reverse direction, the nucleic acids migrate back the same distance as in the first run (arrows with continuous lines) thus refocusing into single bands per slice. Due to the presence of SDS, proteins migrate differently from the first run (arrows with dotted lines) and thus are not refocused. (D) Particle-to-infectivity ratio (P/I) are plotted as a function of the average length of residual nucleic acid. The white and the black circles are from two independent preparations (cf. (118)). The dashed lines are linear regression lines of the experimental values leading to P/I = 1 at ∼50 and 100 nt average length of the nucleic acid. Due to the clearance effect in the animal brain only 4% of the injected infectious particles are present after 20 h and induce further infectivity; consequently, only 4% of the inoculum could be essential for infectivity (for further details see (118)). When the data were corrected for the clearance effect (vertical arrows and continuous lines), 28 and 50 nt average length, respectively, of nucleic acid molecules were present in one infectious unit. Modified from (119) and (118).