Bio-Rad PROTEAN® i12™ IEF System - Manual (Page 46)

PROTEAN i12 IEF System Instruction Manual 42 | 42 |
When planning an IEF experiment, one must
choose between a number of methods for sample
application, two options for IPG strip configuration
(gel-side up or gel-side down), and whether to
use electrode wicks. The PROTEAN
® i12 ™ IEF cell
accommodates all of these options using a single
set of electrodes and focusing trays specific for each
commercially available IPG strip length.
Sample Loading Methods
In the original procedure for IEF on IPG strips
described by Görg et al. (1988), IPG strips were
rehydrated without sample, placed gel-side up for
IEF. The sample was applied in sample cups that
were open at the bottom and pressed against the
gel (cup loading).
A procedure was later described (Rabilloud et al.
1994, Sanchez et al. 1997) in which the sample was
included in the rehydration solution and introduced
uniformly along the IPG strip during rehydration
(in-gel loading). In this approach, sample was diluted
into a volume of rehydration solution appropriate for
the IPG strip length, and the IPG strip was placed
over the sample with its gel-side down. This method
simplified sample application and, in some cases,
improved results, particularly with dilute samples or
larger quantities of sample protein. In-gel sample
loading also allows rehydration and IEF to be
conducted as one continuous unattended operation:
if in-gel sample loading is conducted in the focusing
tray with the electrodes in place and in contact with
the gel, the IEF instrument may be programmed to
start IEF without user intervention following suitable
time for rehydration.
In-gel sample loading may also be conducted under
low voltage (active rehydration). In this technique,
in-gel sample loading is conducted in the focusing
tray under a relatively low voltage (50–100 V). This
can improve entry of high molecular weight proteins.
Despite greater complexity in setup, cup loading is
beneficial in certain circumstances. It generally gives
better results when IEF is conducted on basic pH
gradients (for example, pH 7–10) or when optimum
resolution of basic proteins is desired on wide pH
gradients (Görg et al. 2000, Barry et al. 2003).
Cup loading may also be beneficial for samples
containing high molecular weight or hydrophobic
proteins (Görg et al. 2004). Sample cups should be
placed close to an electrode: positive (+) electrode
placement is recommended for basic gradients, and
negative (–) electrode placement is recommended
for acidic gradients. On wide gradients, the best
resolution is generally observed at the end of the IPG
strip opposite the site of cup placement. Use anodic
placement to improve resolution of basic proteins
and cathodic placement for acidic proteins. Factors
influencing the choice of sample loading method are
summarized in Table B.1.
Appendix B
Sample Loading Methods and
Running Configurations