2-D Gel Electrophoresis: First-Dimension Separation First-Dimension Separation Methods Protocol

2-D Gel Electrophoresis: First-Dimension Separation First-Dimension Separation Methods Protocol
2-D Gel Electrophoresis:
First-Dimension Separation
Protocol
Bulletin 6240
First-Dimension Separation Methods
Master 2-D techniques before proceeding to separate your
own samples with the ReadyPrep™ 2-D starter kit. Premixed
reagents, a standardized sample, and a detailed optimized
protocol allow you to get familiar with 2-D techniques and to
validate your 2-D system.
Protein Load for 2-D Gels
Table 1 shows generally recommended protein loads for 2-D
gels. Because of sample-to-sample variation, the amounts are
a guide only. For narrower pH range IPG strips, more protein
can be loaded, because proteins outside the range of pI
resolution will not remain on the strip to enter the 2-D gel.
For single-pH-unit IPG strips, the amount that can be loaded
can be as much as 4–5 times more, which allows better
detection of low-abundance proteins. For further discussion
of factors related to protein load.
Passive Rehydration With Sample
Passive sample application during rehydration is performed by
placing the IPG strip gel side down in the channel of a focusing
or rehydration tray that contains the sample in an appropriate
rehydration solution. Use the sample volumes given in Table 2.
This procedure will result in rehydration of the strips to their
original thickness of 0.5 mm. Larger or smaller volumes can
be used and the strips will swell to accommodate more liquid
up to a point (Görg et al. 2000). A minimum of 11 hr total
rehydration time is recommended. It is important that the
strips be left in the well for the entire time, even if it appears
that all of the liquid has been absorbed. High MW proteins
cannot enter the gel until the pores are large enough to accept
them, which only occurs when the pores have swelled to their
maximum size.
Table 2. Approximate volumes to hydrate ReadyStrip™ IPG strips.
Table 1. Approximate protein loads for IPG strips.
IPG Strip Length
Analytical Load (Silver or
SYPRO Ruby staining) Preparative Load
(Coomassie staining)
7 cm 10–100 μg protein 200–500 μg protein
11 cm 50–200 μg protein 250–1,000 μg protein
17 cm 100–300 μg protein 1–3 mg protein
IPG Strip Rehydration
Solutions used to rehydrate IPG strips prior to loading a
sample are the same as those used to solubilize or dilute
samples for in-gel rehydration. Methods for rehydration of
strips in buffers (with or without sample) are described in
the following sections.
ReadyStrip Strip Length IPG Volume
7 cm
125 μl
11 cm
200 μl
17 cm
300 μl
If too much solution remains outside the gel in the focusing
tray during electrophoresis, a parallel current path along the
surface of the strip can form in which the proteins will not
be focused. This can result in protein loss and streaking.
To minimize the possibility of a parallel current path, rehydrate
the strips in a disposable rehydration tray, then transfer them
to the focusing tray. During transfer, carefully blot excess liquid
from the strip with moist filter paper prior to beginning the run.
Remove the IPG strip from the protective cover using gloved
hands and forceps. Carefully place the IPG strip in the
rehydration buffer, gel side down, making sure the entire
strip is wetted. There is no “best way” to place dry IPG strips
in contact with solution in the trays. Any of the methods
illustrated in Figure 1 are suitable.
A
B
It is helpful to add a trace of Bromophenol Blue to the sample
solution to observe the hydration process. Allow the liquid to
distribute for about 1 hr before covering the strips with mineral
oil. The IPG strips must be covered to prevent evaporation,
which will cause the urea to precipitate as it becomes more
concentrated. As a precaution against evaporation, mineral
oil should be gently layered on top of each channel until it
completely covers each strip.
Active Rehydration
Fig. 1A and 1B. Strip rehydration method 1. Prop up one of the long edges
of the tray at an angle to the lab bench. Pipet the rehydration solution along
the entire length of the lower corner of each channel (A); place the strip,
edge first, into the liquid (B). Then place the tray flat on the benchtop.
C
D
For active rehydration of IPG strips with sample in a focusing
tray, run the IEF cell under low voltage (50 V). Ensure that
the liquid extends past the electrode wires at each end so
that the entire strip rehydrates and no dry area creates a
discontinuity in the current path. It might be necessary to lift
the ends of the IPG strip slightly to get the liquid to flow to
the ends of the strip. After the sample has been in contact
with the strips for 1 hr, add mineral oil to cover each strip.
The PROTEAN® i12™ IEF cell can be programmed for active
rehydration and to transition automatically into a focusing
run. Alternatively, a pause may be incorporated to allow the
operator to insert a wick under each end of the strip (see the
section below on performing IEF). If this method of sample
application causes a disproportionate ratio of large proteins
to small proteins, try passive rehydration.
Performing IEF
The PROTEAN i12 IEF cell with integrated power supply
and Peltier cooling is recommended for IEF protocols in this
manual. It can simultaneously run up to twelve 11 or 17 cm
IPG strips or up to twenty-four 7 cm strips. Running conditions
can be better controlled by running the same type of sample,
buffer, and IPG strip pH range together.
Positioning Strips and Use of Wicks
Fig. 1C and 1D. Strip rehydration method 2. Pipet the rehydration solution
into the middle of each tray channel (C); bend the strip into a “U” shape and
lower it into the liquid from the center out to the edges (D).
E
Fig. 1E. Strip rehydration method 3.
Pipet the rehydration solution into one
end of each tray channel. Butt the strip
up to the same end of the channel
and lower it into the liquid toward
the opposite end (E).
After the strips have rehydrated, move them to the i12
focusing tray if they were rehydrated in other trays.
Carefully blot excess liquid from the strip with moist filter
paper. Wicks are highly recommended because they collect
salts and other contaminants in the sample. Without wicks,
salts collect at the anode and cathode, producing high
conductivity that can alter the gradient, cause discontinuities
in the gel, and cause “hot spots” or burns. Place a dry wick
on each electrode that is used (Figure 2). Position the wicks
within the indentations of the channels. Pipet 5–8 μl of water
on each wick before positioning the IPG strips.
Fig. 2. Placement of wicks on
the electrodes in each channel
that will be used. IPG strips will
be placed on top of the wicks.
© 2011 Bio-Rad Laboratories, Inc.
Bulletin 6240
Alternatively, if strips are rehydrated in the focusing trays
(either actively or passively), the ends of each strip can be
lifted with forceps and wet wicks inserted between the strip
and the electrodes (Figure 3). Wicks should be wetted but not
soaked. Blot wetted wicks before placing them in the tray.
Voltage Ramping Modes
Voltage ramping can replace traditional stepwise
voltage programming with continuous voltage changes.
The PROTEAN i12 IEF cell (Figure 4) includes three voltage
ramping modes: rapid, linear, and slow. Each ramping mode
is appropriate for the resistance of particular samples.
The combined resistance of the IPG strips, the rehydration
buffer, and the sample determines which ramping mode
should be used. During the focusing process, charged
contaminants move to the electrodes and proteins move to
the pH equal to their pI. While the proteins are being focused,
the resistance of the IPG strip gradually increases until it
reaches a maximum.
Fig. 3. Insertion of wicks under
both ends of an IPG strip
that has been rehydrated in
a focusing tray.
Cover the strip with mineral oil before starting the focusing
run to prevent evaporation and carbon dioxide absorption
during focusing. Channels should be filled nearly to the top
but should not be overflowing. The i12 focusing tray has
rounded corners at both ends of the individual channels that
prevent mineral oil movement into the adjacent channels.
The rounded corners also reduce salt buildup due to
inadequate cleaning between IEF runs. It is important to clean
the focusing trays properly between runs. Channel-to-channel
leakage is common when salts accumulate in the channels.
Focusing Conditions for IPG Strips on the PROTEAN i12 IEF Cell
Table 3 gives suggested total volthours for IPG strip runs.
These conditions are intended as a guide; individual samples
may require more or less time.
Table 3. Broad and narrow ranges.
Start
Find
Voltage Voltage
Volt-Hours Ramp Temperature
ReadyStrip pH 3–10, 3–10 NL, 4 –7, 5 – 8*
7 cm 0V
4,000 V 8–15,000 V-hr 11 cm
0V
8,000 V 20–35,000 V-hr Rapid 20°C
17 cm and 0 V 18 cm
10,000 V 40–60,000 V-hr Rapid 20°C
24 cm 10,000 V 60–80,000 V-hr Rapid 20°C
0V
Rapid 20°C
ReadyStrip pH 3–6 Focusing Conditions**, ***
7 cm 0V
4,000 V 8–10,000 V-hr Rapid 20°C
11 cm 0V
8,000 V 15–20,000 V-hr Rapid 20°C
17 cm and 0 V 18 cm
10,000 V 30–40,000 V-hr Rapid 20°C
24 cm 10,000 V 40–55,000 V-hr Rapid 20°C
0V
ReadyStrip pH
7–10*, †
7 cm 0V
4,000 V 8–16,000 V-hr 11 cm 0V
8,000 V 20–30,000 V-hr Rapid 20°C
17 cm and 0 V 18 cm
10,000 V 40–50,000 V-hr Rapid 20°C
24 cm 10,000 V 60–70,000 V-hr 0V
© 2011 Bio-Rad Laboratories, Inc.
Rapid 20°C
Rapid 20°C
Fig. 4. The PROTEAN i12 IEF cell and accessories.
Each voltage ramping mode controls the rate of voltage
change as follows:
Rapid ramping mode — In rapid ramping mode, salts
and other ionic contaminants are driven from the IPG strips
as rapidly as possible. The limiting factor in reaching
the maximum set voltage is the current limit per strip.
The maximum voltage can be reached in ≤2 hr for highresistance (low-ionic-strength) samples, or in >6 hr for lowresistance samples. In both cases the power supply will
run at the set current limit until a steady state is reached.
This is the mode of choice for many samples, and is
particularly useful to minimize low-resistance sample run time.
Linear ramping mode — In linear ramping mode,
the voltage increases linearly within the programmed time
frame, starting with the final voltage of the previous step
and ending with the maximum voltage programmed.
The resistance of the sample/rehydration buffer system will
determine whether the maximum set voltage can be reached
in the programmed time. This mode is used for samples of
intermediate resistance.
Slow ramping mode — In this mode, the voltage is
increased quadratically:
V = B + (N2 × (E – B)/T2)
where B = starting voltage, E = ending voltage, N = elapsed
time, and T = total time. The run will continue below or at the
current limit. This mode is used for high-resistance sample/
rehydration buffer systems to minimize high power input
initially while achieving high voltage as quickly as possible.
Bulletin 6240
Note: The default current limit in the PROTEAN i12 IEF cell is
50 μA per strip. A higher current limit, up to 99 μA per strip,
can be programmed into a method. All preset methods have
a fixed current limit of 50 μA per strip. In the rapid ramping
mode, the system runs at the set current limit and adjusts the
voltage until the maximum voltage is reached. In the linear or
slow ramping modes, the system follows a specific algorithm
and does not always run at the current limit. The factor that
determines the time needed to reach maximum voltage is the
composition of the sample solution. Systems with high salt
concentration and high sample loads require a long time
to reach steady state. It is not always possible to reach
the maximum set voltage within the programmed time.
High ampholyte concentrations and high protein load also
limit the final attainable voltage.
*The final voltage for each pH range may not be reached, but the total
volt-hours given above are sufficient to properly focus samples with final
voltages as low as 3,000 V (7 cm), 5,000 V (11 cm), and 7,000 V (17 cm,
18 cm, and 24 cm). A lower final voltage will increase total run time.
**The final voltage for this pH range may not be reached, but the total
volt-hours given above are sufficient to properly focus samples with final
voltages as low as 2,000 V (7 cm), 3,000 V (11 cm), and 6,000 V (17 cm,
18 cm, and 24 cm). A lower final voltage will increase total run time.
***Enhanced resolution and separation of proteins may be achieved using
cup loading with sample application at the cathode (–) end of the IPG strip.
†
To ensure success with basic range IPG strips, performing two additional
steps is strongly recommended. The first step is to treat the sample using
the ReadyPrep reduction-alkylation kit (catalog #163-2090). This reduces
streaking caused by disulfide bond formation, which is more problematic
with basic range proteins. The second step is to use cup loading when
loading samples for isoelectric focusing. For more information, refer to the
ReadyPrep reduction-alkylation kit instruction manual (bulletin 4110063).
Storage of IPG Strips After IEF
Because the pH gradient is fixed in the IPG strip gel, focused
proteins are more stable at their pI than in conventional IEF
gels. Focused IPG strips can be stored at –20°C indefinitely
without affecting the final 2-D pattern. IPG strips are bound to
a plastic sheet, so gel cracking, which results from expansion
and contraction during freezing and thawing, is avoided and
the IPG strips retain their original dimensions after thawing.
It is convenient to store IPG strips in rehydration trays or
screwcap plastic tubes, which can then be used to equilibrate
the strips for the second dimension.
This is an excerpt from Bio-Rad’s comprehensive manual, 2-D Electrophoresis for Proteomics (Bulletin 2651).
Bio-Rad
Laboratories, Inc.
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Life Science
Group
Bulletin 6240 Rev A
US/EG
11-0864
1111
Sig 1211
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