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The followmg procedure is adapted from that described by O™Farell et al
(1.5,26). Infected cells are lysed by osmotic shock from added water and three
cycles of freezing and thawing followed by 1 mm of bath somcation at 0°C.
Nucleic acids are then degraded by adding to each sample 0 05 vol of 300 mM
Tris-HCI (pH 7 5) containing 300 mJ4Mg2+, 300 ug/mL DNase 1and 300 &mL
RNase A, and incubating at O°C for 30 min The mixture is then transferred to
dry ice and kept there during the subsequent additions
To 187.5 uL sample is added 80 uL 10% NP-40, 16 p.L ampholmes 3.5-10
(diluted 1:3 m H,O), 20 uL 2-P-mercaptoethanol and 2 16 mg urea When dis-
solved this gives a final volume of approx 400 FL, and the additions can be
adjusted proportionally for other sample volumes. The urea is drssolved by
gentle shaking at room temperature, the mixture put back on dry ice immedi-
ately afterward and stored at -8OOC
Solid samples or pellets are drssolved m an approprtate volume of “lysis
buffer” containing 9.5M urea, 2% (w/v) NP-40, 2% ampholines (1.6% range
pH 3.5-l 0.0 and 0.4% range pH 5.0-7.0) and 5% mercaptoethanol.
Our tubes for the first dimension gels are 10.5 cm long with an inner diam-
eter of 2.3 mm. The solutton for the gel 1smade of 2.75 g urea, 585 uL 30%
acrylamide, 900 pL 10% NP-40, 1170 uL H20, and 250 uL ampholmes pH
3.5-10. Urea is dissolved by gentle shaking at room temperature. At this time
10 uL of 10% ammonium persulfate and 7 pL TEMED are added, and the
solutton is tilled mto the vertical tubes that are sealed m the bottom by pushing
them against parafilm. Enough space should be left at the top for subsequent
loadings. An overlayer of 20 pL H20 is added and the gels allowed to polymer-
ize for approx 1 h When using more basic ampholmes more ammomum
persulfate and TEMED are needed for polymerization.
To keep the gel m position during running, a piece of dialysis tubing is fixed
with a rubber string at the bottom of the tube, avoiding an bubbles. With the
tubes in position m the apparatus for electrophorests the liquid on top of the
polymerized gel is removed and a 10-35 uL sample is loaded per tube. An
overlay of 20 uL 8M urea (stored at -80°C and thawed immediately before
Analysis of HSV Polypepticies 113

use) is added. The extra space in the tube and the upper electrophoresls cham-
ber are filled with 10 tiphosphoric acid. The lower chamber contains 20 rnA4
NaOH. For nonequihbrmm pH gel electrophoresis we run for 3 h at 550 V. The
gels are then pushed gently and either subjected Immediately to electrophore-
US in the second dimension, or put mto glass tubes that are subsequently filled
with “sample buffer” containing 10% (w/v) glycerol, 5% (v/v) mercapto-
ethanol, 2 3% (w/v) ultrapure SDS, and 62.5 MTris-HCl, pH 6.8, then stored
at -80°C. The tube gel IS kept m position on top of the slab gel by pourmg a hot
(80-l OO™C) solution of 1% agarose in “sample buffer.”
Slab gels used for the second dimension are those described by Laemmli
(IO), and need no further descnptlon. A few points, however, should be noted.
A special comb is used to provide a wide slot for the tube gel and a narrow one
for application of a molecular weight marker. We use 5% polyacrylamlde m
the stacking gel. Gradient running gels are allowed to polymerize from the top
by using 2.3 times more ammonium persulfate in the solution with lowest con-
centration of acrylamide (28). Electrophoresis is performed at 35 mA per gel
using bromphenol blue as dye.

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