High performance capillary electrophoresis - T.E.A.M.

Modes
3.4.1 Applications of CIEF
CIEF has been used successfully to measure protein pI and
for the separation of isoforms, and other protein species
problematic by other methods, including immunoglobulins
and hemoglobins, and analysis of dilute biological solutions.
CIEF of protein standards is shown in figure 46. An example
of the use of CIEF for the separation of transferrin isoforms
is shown in figure 47.
Figure 46
CIEF of standard protein mixture 24
Polyacrylamide coated capillary
0.01 AU
pH
mAU
100
75
50
TIFe C
25
Fe N
TIFe C
TIFe C
3 4
2
1
8.6 8.4 8.2
pH 3 – 10 ampholytes
5
1
2
3
4
5
6
7
8
Lentil lectins
Human hemoglobin A (7.5)
Human hemoglobin A (7.1)
Equine myoglobin (7.0)
Equine myoglobin, minor band (6.8)
Human carbonic anhydrase (6.5)
Bovine carbonic anhydrase (6.0)
β - lactoglobulin B (5.1)
6
7
8
Fe N
TIFe C
TIFe C
0
0 3 6 9 12
Time [min]
TI
0 2 4 6 8 10
Time [min]
Mobilization
Figure 47
CIEF of transferrin isoforms 25
Conditions: V = 4kV for focusing and
mobilization, 2 % Bio-Lyte 5/7
(containing 1 mg of transferrin),
L = 14 cm, id =100 mm, l = 280 nm
for protein and 460 for iron
77