Bio-Rad PROTEAN® i12™ IEF System - Manual (Page 47)

Appendix B Sample Loading Methods and Running Configurations
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Table B.1. Advantages and disadvantages of different sample loading methods.
Method Advantages Disdvantages
In-Gel Loading Simple sample application Poor resolution of basic proteins
No precipitation at point of sample application
Accommodates dilute samples and larger protein loads
Passive Focusing can follow rehydration without manual intervention Not all proteins, particularly large or
if performed within the IEF instrument hydrophobic proteins, will be taken up
Active More effective with certain proteins, particularly those of high Rehydration must occur within the IEF
molecular weight instrument
Cup Loading More effective for basic proteins Setup more complicated; the cup must
form a seal with the IPG strip
Can improve resolution at extremes of the pH gradient High protein loads are difficult to
(the end opposite the point of application) accommodate; concentrated samples
are required
Sample precipitation may occur at the
point of application
IPG Strip Configuration
The orientation (gel-side up or gel-side down) of
the IPG strip during IEF is largely determined by the
sample loading method employed:
■
Cup loading requires gel-side up strip placement
so that the sample cup may be placed in contact
with the gel surface
■
In-gel sample loading is conducted gel-side down.
If the IEF cell is programmed for an unattended
start following rehydration, IEF must be conducted
gel-side down as well
■
If in-gel sample loading is performed in the
rehydration/equilibration tray, IEF may be
performed either gel-side up or gel-side down.
This is largely a matter of user preference, though
improved resolution may be observed with the gel-
side up, particularly with higher protein loads
Electrode Wicks
Electrode wicks serve as a sink for ionic sample
contaminants and proteins with pIs outside the pH
range of the IPG strip used. They also prevent drying
of the ends of the IPG strips during IEF. Electrode
wicks may be placed between an electrode and
the IPG strip in either running configuration (with
the PROTEAN i12 IEF cell, specific electrode wicks
are provided for each configuration). Rehydration
in the focusing tray cannot be performed with
electrode wicks in place. In-gel sample loading with
an unattended IEF start, therefore, precludes the
use of electrode wicks. However, a pause may be
programmed following rehydration during which
electrode wicks may be inserted. In many cases, the
use of electrode wicks has little effect on separation
quality, and they may be omitted for convenience in
either running configuration if satisfactory results are
obtained in their absence.