Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 21)

Table 3.1. Advantages and disadvantages of sample loading methods.
Method
Advantages Disadvantages
In-gel rehydration Simple sample application Poor resolution of basic proteins
No precipitation at the point of sample application
Accommodates dilute samples and larger protein loads
Passive Focusing can follow rehydration without manual Not all proteins, particularly large or hydrophobic
intervention if performed within the IEF instrument proteins, will be taken up
Active More effective with certain proteins, particularly those Rehydration must occur within the IEF instrument
of high molecular weight
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 accommodate;
(the end opposite the point of application) concentrated samples are required.
Sample precipitation may occur at the point
of application
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2-D Electrophoresis Guide Theory and Product Selection Chapter 3: The First Dimension: Isoelectric Focusing (IEF)
The composition of the rehydration solution should
also resemble the composition of the sample solution
in terms of the additives present to aid solubility. If the
sample was prepared using thiourea, the rehydration
solution should also contain thiourea. Likewise, the
same detergent should be used. Otherwise, transition
from one solution to the other can cause precipitation
of proteins that are soluble in the sample solution but
not in the rehydration solution
Sample Application during Rehydration
In this option, the sample is prepared in, or diluted into,
rehydration solution and introduced to the IPG strip
at the time of rehydration. As the strips hydrate for
at least 12 hr, proteins in the sample are absorbed
and distributed over the entire length of the strip
(Rabilloud et al. 1994, Sanchez et al. 1997).
Sample application can be in either the absence
(passive) or presence (active) of applied voltage:
■ ■
Active rehydration is performed in the IEF cell. A low
voltage (30–100 V) is applied, and proteins enter the
gel matrix under current as well as by absorption.
Active rehydration with the sample is believed to
promote the entry of large proteins into the strip by
applying an electrical “pull”
■ ■
In passive rehydration, proteins enter the gel
by absorption only. This allows efficient use of
equipment, since strips can be rehydrated in sample
rehydration trays while other samples are focused in
the IEF cell
Sample Application
Commercial IPG strips are dehydrated and must be
rehydrated to their original gel thickness (0.5 mm)
before use. The protein sample can be applied
to the IPG strip either during or after rehydration,
and rehydration can be done in either disposable
rehydration/equilibration trays or directly in the
focusing tray. Sample application during rehydration
is the easiest and, in most cases, most efficient way to
apply sample. In some instances, however, it is best
to rehydrate the IPG strips and then apply sample
through sample cups while current is applied
(cup loading) (Table 3.1). Each method is discussed
in the following sections.
The rehydration solution generally contains the
following components to maintain protein solubility and
allow tracking of the separation (see Part II, Methods
for recipes):
■ ■
Chaotrope — urea (8 M or up to 9.8 M if necessary
for sample solubility), with or without 2 M thiourea
■ ■
Detergent — nonionic or zwitterionic detergent such
as CHAPS, Triton X-100, or NP-40 at 0.5–4% (w/v)
■ ■
Reducing agent — 20–100 mM DTT
■ ■
Ampholytes — 0.2% (w/v), usually pH 3–10;
concentrations up to 1% (w/v) may be used,
though this reduces the voltage and results in
correspondingly longer runs
■ ■
Tracking dye — a trace of bromophenol blue to
render the IPG strip more visible for simplified handling
and act as a tracking dye for confirmation of focusing
Setup for IEF
For IEF, the rehydrated IPG strips are placed into the
focusing tray. The orientation (gel-side up or gel-side
down) of the IPG strip in the focusing tray 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
configuration, particularly with higher protein loads
In addition, electrode wicks may be placed between
the electrode in the focusing tray and the IPG strip in
either running configuration. 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. In some cases, however, 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.
Whether IPG strips are rehydrated actively or passively,
they must be incubated with sample for at least 12 hr
prior to IEF. This gives high molecular weight proteins
time to enter the gel after the gel has become fully
hydrated and the pores have attained full size.
Sample application during rehydration works
because IEF is a steady-state technique; therefore,
proteins migrate to their pI independently of their initial
positions. The advantages of these approaches
over cup loading are:
■ ■
Simplicity
■ ■
Reduced risk of sample precipitation, which can
occur with cup loading at the sample application
point if sample concentration is too high
(Rabilloud 1999)
■ ■
Shorter focusing times can be used because the
sample proteins are in the IPG strip prior to IEF
■ ■
Large amounts of protein can be loaded
Sample Application by Cup Loading
To apply samples after IPG strip rehydration,
the sample is loaded into sample cups positioned
on the rehydrated strip. This technique can be more
challenging than in-gel sample loading from a technical
standpoint, but it can be beneficial in the following
cases (Cordwell et al. 1997, Görg et al. 2000):
■ ■
When samples contain high levels of DNA, RNA,
or other large molecules, such as cellulose
■ ■
When running acidic and basic IPG strips;
for example, pH 7–10
■ ■
When running micro-range IPG strips spanning
~1 pH unit
■ ■
For samples that contain large amounts
of glycoproteins