4
Method (Column): C-Terminal
5.0 (Cycle 1)
Total time: 88.7 min
Step 1: Cycle 1 begins with an extended dry to remove any residual solvent
from the sample loading process. There
is also a pause to provide time for
proper HPLC column equilibration after
switching from N-terminal sequencing
Steps 3, 25, 47, 72: The volume of
R3 delivered during this step should
be just enough to wet the membrane.
Steps 7, 13, 19, 22, 29, 35, 41, 44,
51, 57, 63, 66, 69,76, 79, 82, 88,
91, 109, 112: The delivery of S2/3
and S3 should completely wet the
Zitex membrane. The solvent deliveries are held against a closed valve,
enabling 1/2 to 3/4 of the membranecontaining column compartment
(bottom) to remain filled with solvent for the duration of the step.
Steps 10, 32, 54: The delivery of 1R
should completely wet the membrane, but not flood it. The initial
delivered volume (about 1/2 the volume of the column) should blow
through the column wetting the Zitex
strip. The 1R reagent delivery is then
held against a closed valve, enabling
only a small volume of 1R to sit at the
bottom of the column (approximately 1 slit distance as measured by the
slits on the column wings).
Steps 16, 38, 60: The delivery of
2R should completely wet the membrane. The initial delivered volume
(about 1/2 the volume of the column)
should blow through the column wetting the Zitex strip. The 2R reagent
delivery is then held against a closed
valve, enabling only a small volume
of 2R to sit at the bottom of the column (approximately 1 slit distance
as measured by the slits on the column wings).
Step Description Primary Temperature
Time
1: Activation: Dry column DOWN and wait 120.0 60
2: Activation: Meter R3 7.0 60
3: Activation: Deliver R3 DOWN 30.0 60
4: Wash: Flush RV2 with S3 5.0 80
5: Activation: Dry column DOWN 50.0 80
6: Wash: Meter S2/3 9.0 80
7: Wash: Deliver S2/3 DOWN (closed) 15.0 80
8: Wash: Dry column DOWN 5.0 80
9: Couple: Meter 1R 5.0 80
10: Couple: Deliver 1R UP 30.0 80
11: Couple: Dry column DOWN 10.0 80
12: Wash: Meter S2/3 and S2A 9.0 80
13: Wash: Deliver S2/3 DOWN (closed) 15.0 80
14: Wash: Dry column DOWN 5.0 80
15: Couple: Meter 2R 5.0 80
16: Couple: Deliver 2R UP 10.0 80
17: Couple: Dry column DOWN 5.0 80
18: Wash: Meter S2/3 and S2A 9.0 80
19: Wash: Deliver S2/3 DOWN (closed) 15.0 80
20: Wash: Dry column DOWN 5.0 80
21: Wash: Meter S2/3 9.0 80
22: Wash: Deliver S2/3 DOWN (closed) 15.0 80
23: Wash: Dry column DOWN 20.0 80
24: Activation: Meter R3 7.0 60
25: Activation: Deliver R3 DOWN 30.0 60
26: Wash: Flush RV2 with S3 5.0 80
27: Activation: Dry column DOWN 50.0 80
28: Wash: Meter S2/3 9.0 80
29: Wash: Deliver S2/3 DOWN (closed) 15.0 80
30: Wash: Dry column DOWN 5.0 80
31: Couple: Meter 1R 5.0 80
32: Couple: Deliver 1R UP 30.0 80
33: Couple: Dry column DOWN 10.0 80
34: Wash: Meter S2/3 and S2A 9.0 80
35: Wash: Deliver S2/3 DOWN (closed) 15.0 80
36: Wash: Dry column DOWN 5.0 80
37: Couple: Meter 2R 5.0 80
38: Couple: Deliver 2R UP 10.0 80
39: Couple: Dry column DOWN 5.0 80
40: Wash: Meter S2/3 and S2A 9.0 80
41: Wash: Deliver S2/3 DOWN (closed) 15.0 80
42: Wash: Dry column DOWN 5.0 80
43: Wash: Meter S2/3 9.0 80
44: Wash: Deliver S2/3 DOWN (closed) 15.0 80
45: Wash: Dry column DOWN 20.0 80
46: Activation: Meter R3 7.0 60
47: Activation: Deliver R3 DOWN 30.0 60
48: Wash: Flush RV2 with S3 5.0 80
49: Activation: Dry column DOWN 50.0 80
50: Wash: Meter S2/3 9.0 80
51: Wash: Deliver S2/3 DOWN (closed) 15.0 80
52: Wash: Dry column DOWN 5.0 80
53: Couple: Meter 1R 5.0 80
54: Couple: Deliver 1R UP 30.0 80
55: Couple: Dry column DOWN 10.0 80
56: Wash: Meter S2/3 and S2A 9.0 80