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Using CD 1
Using CD 1
Using CD 1
where
nTRP,
nTYR,
and
nS-‐S
are
the
number
of
TRP
residues,
TYR
residues,
and
disulfide
bonds
in
the
protein,
respectively.
• Measure
the
absorption
at
280
nm
of
the
protein
solution
in
20
mM
phosphate
buffer,
pH
6.5
with
6
M
guanidine-‐HCl.
Ensuring
that
the
total
absorption
at
280
nm
is
between
0.1
and
1.0
where
the
absorption
is
linear.
Typical
CD
spectra
of
alpha
helix,
beta
sheet,
and
random
coil
Starting
the
instrument
1. Turn
on
N2
gas
and
adjust
regulator
to
provide
7
lpm
flow
rate.
a. NOTE:
At
7
lpm
approximately
1,000
kPa
are
depleted
in
one
hour.
b. NOTE:
Nitrogen
is
cheap,
$5.00
per
tank,
don’t
push
the
tank
to
the
end
c. NOTE:
You
can
switch
N2
tanks
while
running
the
CD
but
act
quickly.
Shutdown
the
instrument
if
something
goes
wrong
and
you
cannot
restore
the
N2
purge
gas
within
a
few
minutes.
d. NOTE:
Running
the
CD
for
any
significant
length
of
time
(longer
than
5
minutes)
without
a
nitrogen
purge
will
damage
the
optics
and
will
result
in
up
to
a
$5,000
repair.
2. Turn
on
the
cooling
water
bath
a. Do
not
change
the
set
temperature
b. NOTE:
This
does
not
control
the
sample
temperature
3. Wait
20
minutes
to
completely
purge
the
system
with
N2
gas
4. Turn
on
the
orange
power
switch
and
wait
10
seconds
5. Turn
on
the
green
power
switch
6. Log
the
starting
lamp
hours
in
the
logbook.
The
timer
is
located
on
the
power
supply.
7. Turn
on
the
temperature
controller
and
hit
the
START
button.
a. Do
not
touch
other
buttons.
8. Start
the
“Spectra
Manager”
software
9. Set
the
sample
temperature
(see
the
next
section
for
details)
10. Allow
20
minutes
for
instrument
to
properly
warm-‐up.
11. Measure
the
photomultiplier
voltage
at
300
nm
with
an
empty
sample
chamber
(including
the
spacer)
and
a
1
nm
bandwidth
and
record
the
voltage
in
the
logbook.
Setting
sample
temperature
1. Go
to
Measurement
à
Accessory
à
Temperature
and
select
Jasco
Peltier
2. Go
to
Control
à
Accessory
à
Enter
desired
temperature
à
Apply
Shutting
down
the
instrument
1. Exit
the
“measurement”
program
that
you
were
running
2. In
“Spectra
Manager”
shutdown
the
instrument
by
going
to
Instrument
à
Stop
and
then
close
the
“Spectra
Manager”
3. Turn
off
the
temperature
controller
4. Turn
off
the
water
bath
5. Turn
off
the
green
power
switch
on
the
power
supply
6. Turn
off
the
orange
power
switch
on
the
power
supply
7. Record
the
ending
lamp
hours
in
the
logbook
8. Turn
off
the
N2
gas