Bio-Rad PROTEAN® i12™ IEF System - Manual (Page 49)

PROTEAN i12 IEF System Instruction Manual | 45 | 45
Appendix C IEF Protocols
Note, however, that IEF is performed under current-
limited conditions, so the maximum programmed
voltage may never be reached, depending on the
programmed voltage, the nature of the sample, and
the intrinsic resistance of the IPG strip used.
Since the duration of the prolonged focusing phase
is specified in V-hr, the actual duration depends on
the average voltage during focusing. Focusing may
conclude at different times for IPG strips run at the
same time with the same protocol. It is, therefore,
important to include a hold step during which the
IPG strip is held at a relatively low voltage to maintain
focusing until the IPG strip can be removed from the
instrument.
Table C.1. Recommended focusing and hold voltages.
Voltage (V)
IPG Strip (cm) Focusing Hold
7 4,000 500
11 8,000 750
17 10,000 1,000
18 10,000 1,000
24 10,000 1,500
Fig. C.1. Voltage profiles in a two-step protocol. The ramping mode
for step 2 is either rapid, linear, or gradual. Step 1 is 500 V for 1 hr, step 2
is 1,000 V for 1 hr, and current is non-limiting.