. 14
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arranged in a parallel fashion forming a fibril27-34 nm in diameter and some-
times consisted of four filaments at one end and two pairs of filaments forked
at the other end. There appeared to be a great deal of variability in the way
these individual filaments interacted so intermediate forms were also observed.
Typical configurations of ovine SAF and BSE tibrils using the described pro-
cedures are shown in Fig. 2A,B. Similar tibrils detected in brain extracts from
a domestic cat and a greater kudu are shown in Fig. 2C,D.
Fig. 2. Electron micrographs of fibrils from four different species obtained using the
n-lauroylsarcosine extraction and proteinase K digestion technique followed by stain-
ing with phosphotungstic acid to provide negative contrast. (A) This micrograph shows
the diverse nature of SAF, as found in extracted ovine brain tissue. The Type I struc-
tures (large arrows) have a “bendy” appearance but also present are the brittle looking
fibrils (small tailed arrows), parallel forms (P), and short forms (S). Barline = 100 nm.
(B) Micrograph of a cluster of extracted BSE tibrils showing predominately Type I
configurations that are closely grouped. Residual n-lauroylsarcosine detergent can be
clearly identified by its characteristic striped appearance (Is). Ferritin particles (fe) are
often seen in brain tissue preparations after biochemical extraction. Barline = 100 nm.
Electron micrographs of fib& from four different species obtained using the n-lauroyl-
sarcosine extraction and proteinase K digestion technique followed by staining with
phosphotungstic acid to provide negative contrast. (C) Micrograph showing predomi-
nately Type I tibrils extracted from the brain tissue of a domestic cat. (continued)
Diagnosis of BSE and Scrap/e 95

Results are designated as follows:
1 Positive. Identification of undisputed tibrils of any type within the definlt1ot1 of
those described 1n the original publlcatlon of SAF (2).
2 Negative: No fibrils fitting the detinitlon of SAF (2) found after completing a
search of 20 grid squares in a 20-min period
3. Inconclusive Where on rare occasions a sample cannot be classlfled into either
positive or negative a further grid or grids should be prepared for examination
either from the same extract or an extract from another part of central nervous
tissue Examine as determined and reclassify.
3.3. Extraction and Purification of Prpc for lmmunoblotting
In outline, the method consists of extracting and purifying the abnormal,
tibr11 form of PrP from brain or cervical spmal cord (26), analyzing the purt-
tied protein by polyacrylamlde gel electrophoresls (PAGE) (28), followed by
Western blotting (29).
All centrifugatlons are carried out using a Beckman TLlOO benchtop ultra-
centrifuge with a 100.3 fixed angle rotor and thick walled polycarbonate cen-
trifuge tubes (max speed of rotor 100,000 rpm giving a g max figure of
54O,OOOg), using a Beckman L8-60M floor standing ultracentrifuge with a
70T1 fixed angle rotor (max speed of rotor 60,000 rpm g1vmg a g max figure of
37 1,OOOg).Diagrammatic representations of each of the two stagesof the pro-
cedure are shown in Fig. 3A,B.
3 3.7. Stage l--Detergent Extraction Procedure for PrP (Fig. 34
1 Take 4 g of brain or cervical spinal cord
2. Cut into small pieces and add 5 mL of BLB with 10 yL of 100 mM PMSF and 10 pL
of 100 mMNEM
3. Homogenize 1n a sealed unit laboratory mixer (e.g., Sllverson) for 30 s.
4 Add another 5 mL of BLB and rehomogenize 1f necessary
5 Centrifuge at either:
a. 20,OOOg average (17,000 rev/mm) for 30 min at 10°C 1n a 70-T1 Beckman
ultracentrifuge rotor; or
b 22,000g average (20,000 rev/min) for 10 m1n at 10°C 1n a Beckman TL 100
bench ultracentrifuge
6. Carefully tip the supernatant into clean centrifuge tubes and centrifuge at either
a. 177,000g average (46,000 rev/mln) for 2 h 30 min at 10°C 1na 70 T1 Beckman
ultracentrifuge rotor, or

The detergent residues may appear as oval or in strands (Is). (D) Micrograph of tibrils
extracted from a greater kudu (Tragelaphus strepsiceros). N-lauroylsarcosine deter-
gent residue is again apparent (1s) and 1n this micrograph ferritln particles can be seen
1n abundance (fe). Ferrltin particles have been observed 1n extracts from positive and
negative animals from all species.
Stack, Keyes, and Scott
A 4 g I Fresh unfixed brain or spinal card

Homogenise in 1Oml BLB for 30 seconds

\y .Centdfuge
22,OOOgl 10 min-


<˜: A
\ Centrifuge *
54o,ooog/ 20 mln

J supernatant
Resuspend in 3ml distilled water
plus 501˜1 1M TrislHCl pH 7.4

Add Bml
15%KIHSB l-l

U -&-----&

3ml t, 3ml t, Sml
Stir at 37oc
stir at 37oc
30 min
30 min

B Split into two x 4.5ml Aliquotr


540,OOOd 25 min

-..---+ Drain tubes carefully
Leave upright overnight

Discard nd pellet in 50111 distilled water
supematant &Jpt and continue with
Western Blotting Analysis

Fig. 3. Extraction method for disease-specific PrP detection by Western blotting.
(A) Stage l-detergent extraction. (B) Stage 2-proteinase K digestion. *Asterisk
indicates Beckman TL 100 centrifuge with TL 100.3 fixed angle rotor.
Diagnosis of BSE and Scrap/e 97
b 540,OOOg average ( 100,000 rev/mm) for 20 mm at 10°C m a Beckman TL 100
bench ultracentrtfuge
7 Discard the supernatant and take up the pellet in 3 mL dtstilled water with 50 uL
lMTris-HCl, pH 7 4 (0 0167&4), by gentle aspiration wtth a ptpet
8 Incubate in a waterbath at 37°C for 30 min while stirrmg
9 Add 6 mL of 15% KI-HSB and incubate for a further 30 mm as m step 8
10. Divide the mixture above mto two ahquots of 4.5 mL
3.3.2. Stage 2-Protemase K Digestion
and Purification of PrP (Fig 36)
1 To one ahquot (from step 10 above) add 45 uL of 1 mg/mL PK and Incubate for
I h as m step 8.
2 To the other 4 5 mL add 4 5 mL of 10% KI-HSB and then either
a Overlay the total onto 2 mL of 20% sucrose and centrifuge at 189,OOOg aver-
age (51,000 rev/mm) for 1 h 30 mm at 10°C m a 70 Ti Beckman ultracentri-
fuge rotor; or
b Divide into 4 x 2 25 mL ahquots and overlay each onto 0 5 mL of 20% sucrose
and centrifuge at 540,OOOg average (100,000 rev/min) for 25 mm at 10°C m a
Beckman TLlOO bench ultracentrtfuge.
13 Carry out step 12 on the PK-treated sample (from step 11)
14 Carefully tip off supernatants and dram the tubes well
15 Leave tubes standing upright overmght (or 2-3 h) to allow the pellet to dry a little
(do not leave inverted because pellet may become detached)
16 Take up the pellet m a small (approx 50 pL) amount of distilled water, by gentle
aspnation with a micropipet
Samples now can be treated in the appropriate manner and analyzed by poly-
acrylamide gel electrophorests and Western blotting.
3.4. Polyacrylamide Gel Electrophoresis (PAGE) and Western
lmmunoblotfing (Fig. 4A, 6)
Methods vary with different electrophoresls systems.The method described
here applies to the large format (18 x 15 cm) BRL vertical gel electrophorests
system and the BloRad (Richmond, CA) TransBlot cell For different equip-
ment, the manufacturer™s instruction manuals should be followed. Note:
Minigel systems that economrze on reagents are available, but so far the sys-
tem described herem has shown better resolution.
3.4.1. Procedure for PAGE and Western lmmunoblotting
1 Wash, rmse, and dry the glass electrophorests plates ensuring that they are free of
all grease and detergents
2 Wipe the inside surfaces of the plates with acetone
3 Ltghtly grease the 1 5-mm thick spacers with petroleum Jelly (Vaseline) and
assemble the plates, clippmg firmly together.
98 Stack, Keyes, and Scott

Fig. 4. Immunoblots of brain material from negative and BSE affected animals.
(A) Positive antiserum (+veAS) tested against negative (-ve) and positive (+ve)
BSE brain material, with either proteinase K (PK) or non-proteinase K (non PK) treat-
ment. Immunoblots of brain material from negative and BSE affected animals. (B)
Negative antiserum (-veAS) tested against negative (-ve) and positive (+ve) BSE brain
material, with either proteinase K (PK) or non-proteinase K (non PK) treatment. Far
left-hand lane of each immunoblot shows a range of molecular weight markers.

4. Level the plates with a spirit level.
5. Measure down approx 3.5 cm from the top of the smallest glass plate and make a
mark. (This will be the top of the separating gel-leaving room for a stacking gel.)
6. Make up the 12% acrylamide separating gel as indicated in Section Vol-
umes are enough for one gel plate.
7. Mix gently and degas using a vacuum pump for l-2 min. (Acrylamide will not
set in the presence of oxygen.)
8. Pour or pipet the gel between the glass plates to approx 2 mm above the marks
made earlier (gel will shrink slightly on polymerization).
Dlagnosls of BSE and Scrap/e
9 Carefully ptpet a layer of dtsttlled water on top of the gel (Allow the gel to set
but not m strong sunlight.) When the gel has set (approx 45 min) a sharp lme IS
vlstble between the gel/water layer
10. Make up the stackmg gel (see Section 2.1 2.3.)
11 Mix and degas as in step 7
12. Pour the water from the top of the separatmg gel Add the stackmg gel, wrth a
ptpet, to completely fill the unoccupted space
13. Place the 20-well “comb” mto the stacking gel, ensurmg that no bubbles are
trapped under the teeth Allow to set The outstde wells should not be used
because of edge effects, where “smiling” will be seen Four wells/sample ˜111
be needed, plus two wells for the molecular weight markers Therefore, four
samples can be analyzed on each gel plate. Gels either can be used munedtately or
covered with damp tissue and kept overnight at room temperature for use the fol-
lowmg day
14 Make up the electrode buffer and pour tt carefully mto the lower reservou of the
electrophoresrs tank (prpet out any froth that may occur)
15 Remove the clamps from around the gel/glass plate assembly and remove the
lower spacer
16 Place the assembly into the lower buffer, ensurmg that no an IS left m contact
with the lower edge of the gel.
17 Clamp the assembly to the top reservoir of the tank and fill wtth electrode buffer
Check for leaks.
18 Carefully remove the comb from the gel
19. MIX the pellet m disttlled water (from step number 16 of the PrP27-30 extractron
and purlficatron procedure) with an equal volume of disruptron buffer
20 Botl for 6 mm
21. Load 10-50 uL of sample into each well as appropriate. PK and non-PK treated
samples should be tested against positive and negative anttsera so that four wells/
sample wrll be needed (each bemg loaded with the eqmvalent of approx 1 g of
ortginal brain maternal).
22. Load a prestained high range protein molecular weight marker (Gtbco-BRL
[Gaithersburg, MD] cat no 560-604lLA), at one end of each batch of sam-
ples so that tt IS easy to cut the membrane m the correct place after Western
23 Connect the tank to an appropriate power supply and run at a constant current of
30 mA/gel plate until the samples reach the stacking/separating gel interface
(approx 1 h)
24. Turn the current to 40 mA/gel and continue electrophoresis until the blue dye
front approaches the bottom of the separating gel (approx 4 h) Before the elec-
trophoresis is complete
25. Make up the blotting buffer and set up 1 blotting cassette/gel as described m
steps 26-28.
26 Fill a large tray with blotting buffer and lay the black side of an open cassette mto
it (ensure the hinges are on the outstde of the cassette).
Stack, Keyes, and Scott
21 Lay one piece of White “Scotchbrrte” (BioRad fiber pads) onto each half of the
cassette and onto each of those place two pieces of chromatography paper
(Whatman [Matdstone, UK] 1 Chr) cut to the size of the “Scotchbrtte ”
28 Cut a piece of transfer membrane to the size of the gel and “wet” according to the
manufacturer™s spectficattons Immobtlon IS currently used, which needs wetting
with methanol and then soaking m distilled water and blottmg buffer. When elec-
trophorests from step 24 IS complete.
29. Turn off the power supply and remove glass plate assembly from the tank
30 Pry apart the glass plates and cut away the stacking gel
31 Remove the gel and lay onto the chromatography paper (on the black half of the
cassette) Smooth over to ensure that no bubbles are trapped between any of the layers
32 Lay the “wetted” transfer membrane onto the gel and again smooth over (Pro-
teins will not transfer where there are an bubbles )
33 Lay on the other two layers of chromatography paper and “Scotchbrrte,” smooth
over, and close the cassette There should now be a “sandwrch” consrstmg of the
following layers:


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( 45 .)