1.
Buffer and Sample Preparation.
a.
Buffer “A”: Empty the contents of the bottle labeled Buffer “A” into a 500 ml graduated cylinder. Add degassed, deion-
ized water to 500 ml volume. Stir briefly. Label container as Buffer “A”.
b.
Buffer “B”: Prepare Buffer “B” and label container as described above.
c.
Sample: Dissolve the anion exchange standard in Buffer “A” using the syringe to measure 6.5 ml of buffer into the bottle
of protein standards. Replace the stopper and shake gently to dissolve.
2.
Press the Program mode key; select New Method.
3.
Select Time programming mode.
4.
Program the Pump.
a.
Press ADD.
b.
Select Buffer “A” with the Previous/Next keys; press OK.
c.
Enter the step length of 1 minutes; press OK.
d.
Enter flow rate of 3.0 ml/min; press OK.
e.
You have now entered the first step of the method (Buffer “A”, step length of 1 minute, flow rate of 3.0 ml/min.) Enter the
remaining steps.
Step 2. Gradient 0% to 50% “B”, step length 5 minutes, flow rate 3.0 ml/minute.
Step 3. Buffer “B”, step length 3 minutes, flow rate 3.0 ml/minute.
Step 4. Buffer “A”, step length 3 minutes, flow rate 3.0 ml/minute.
f.
After entering Step 4; press OK.
5.
Set the Alarm.
a.
Press the Alarm soft key.
b.
Press ADD.
c.
Enter a time of 1 minute for Alarm 1 and check that Hold Methods is set to NO.
d.
Press OK twice.
The alarm sounds after 1 minute to remind you to turn the valve back to the load position. If left in the Inject position, the
gradient will flow through the loop first instead of going directly to the column.
6.
Enter the fraction collector program.
a.
Press the Frac Coll soft key.
b.
Select ALL.
c.
Enter a fraction size of 0.5 minute.
d.
Press OK twice, then select DONE.
e.
Press SAVE, name the method “DEMO 1”, and press DONE.
7.
Load Sample loop with protein standard.
a.
Turn the MV-6 Injector valve knob counter-clockwise as far as it will go.
b.
Draw 2 ml of the protein standard mixture into the syringe.
c.
Insert the syringe in the top port of the MV-6 injector valve, and fill the sample loop. Leave the syringe in the port when
you have injected the sample — this prevents the sample from siphoning out of the loop.
8.
Start Method.
a.
Press the Run mode key. The system will count down 10 seconds, then start the method.
b.
When the method starts, turn the injector valve knob to the right as far as it will go. When the alarm sounds, turn the valve
knob to the left.
c.
If you are using a Bio-Rad 2128 fraction collector, press “Park”, then “Yes” when method is complete.
-0.50
0
4
6
8
10
12
1
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
0
10
20
30
40
50
60
70
80
90
100
Column: Econo Pac High Q Cartridge (Catalog #732-0028)
Buffer A: 25 mM Tris HCI, pH 8.1
Buffer B: 25 mM Tris HCI, pH 8.1 + 0.5 M NaCI
Standard: Anion Exchange Std. (Catalog #125-0561)
Equine myoglobin is not retained on the column —
it elutes in the void volume (first peak). The remaining
three proteins bind to the column and elute separately
as the salt concentration increases.
A.U x1.E-2
mS/cm
TIME (MINUTES)
RUN CONDITIONS
2
3
4
5
6
7
8
9
10
11
12
E
s
Life Science
Group
Web site
www.bio-rad.com
USA
(800) 4BIORAD
Australia
02 9914 2800
Austria
(01)-877 89 01
Belgium
09-385 55 11
Brazil
55 21 507 6191
Canada
China
86-10-8201-1366/68
Denmark
Finland
France
01 47 95 69 65
089 318 84-177
Hong Kong
852-2789-3300
India
(91-124) 6398112/113/114
Israel
03 951 4124
Italy
34 91 590 5200
Japan
03-5811-6270
Korea
82-2-3473-4460
Latin America
305-894-5950
Mexico
52 5 534 2552 to 54
The Netherlands
New Zealand
64-9-4152280
47-23-38-41-30
Russia
7 095 979 98 00
Singapore
65-2729877
Spain
34-91-590-5200
Sweden
46 (0)8-55 51 27 00
Switzerland
061-717-9555
United Kingdom
0800-181134
00-000 0000 Sig 0101
Bulletin 0000 US/EG Rev A
Bio-Rad
Laboratories, Inc.
4106528 Rev A
