Bio-Rad PROTEAN® i12™ IEF System - Manual (Page 48)

PROTEAN i12 IEF System Instruction Manual 44 | 44 |
Optimum IEF conditions depend on the composition
and complexity of the sample and on the pH range
and length of the IPG strip. The PROTEAN
® i12 ™ IEF
cell has preprogrammed protocols for each length
and pH range that can suit most circumstances
and that also serve as convenient starting points for
optimization.
IEF Protocols
IEF should begin under a gradual increase in voltage
followed by a prolonged focusing phase at the
maximum voltage advisable for the IPG strip length
used. Focusing occurs until a set number of Volt-
hours (V-hr) have accumulated.
Optimal duration (in V-hr) depends on the length of
the IPG strip and the pH gradient. Current is limited
at a recommended 50 µA per IPG strip (though up
to 100 µA is possible), and the resistance of the IPG
strip increases over the course of IEF as ions are
depleted from the IPG strip. Ohm’s law
1 dictates that
when current is held constant, voltage increases as
resistance increases. Voltage, therefore, increases
of its own accord from its initial low value over the
course of the run.
The individual lane control provided by the
PROTEAN i12 IEF cell ensures that the current limit is
not exceeded in any IPG strip, even in situations
1 V = IR, where V=voltage, I=current and R=resistance.
where conductivity differs significantly among
samples run at the same time. A one-step protocol
is adequate in most circumstances, as the voltage
will rise gradually without need for a phased protocol
with programmed voltage ramping. A gradual
protocol, however, may be used with heavy protein
loads or when high levels of charged contaminants
are present. Both rapid (single-step, “R”) and gradual
(ramped, multistep, “G”) protocols are provided
for most IPG strip types. Gradual focusing is
recommended for micro-range IPG strips, so only
gradual preprogrammed protocols are provided for
narrow-range IPG strips.
The PROTEAN i12 IEF cell has three ramping modes
for each step in a focusing protocol (Figure C.1):
■
Rapid ramping mode — the voltage limit is kept
constant throughout the protocol step. The voltage
limit changes abruptly as the protocol transitions
from one step to the next
■
Linear ramping mode — the voltage limit
increases linearly within the programmed time
frame, starting with the final voltage of the previous
step and ending with the programmed voltage for
the current step
■
Gradual ramping mode — the voltage limit is
increased quadratically according to:
V = B + (N
2 × (E – B)/T 2 ), where B = starting
voltage, E = ending voltage, N = elapsed time, and
T = total time
Appendix C
IEF Protocols