Westermeier R., Naven T., H?pker H.-R. Proteomics in Practice: A Guide to Successful Experimental Design

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1 Electrophoretic Techniques80

Fig. 1.23: Rehydration cassette with 0.5 mm gasket. Note the

protruding ends of the U-shaped film added to achieve 0.5 mm

thick gels on film supports.

Reswelling tray The disadvantages of the cassette technique are

avoided when using a reswelling tray (see Figure 1.24), as suggested

by Sanchez et al. (1997). The liquid volume offered to the strips must

be exactly controlled:

If the liquid volume is too big, the strip will pre-

ferably reswell with the low molecular weight

compounds and leave compounds with higher

viscosity and higher molecular weights outside.

There would not be any control over the reagent

concentrations inside the strip.

If the liquid volume is too small, the resulting

pore size will be too small to allow high molecu-

lar weight proteins to enter the gel.

Sanchez J-C, Rouge V, Pisteur

M, Ravier F, Tonella L, Moos-

mayer M, Wilkins MR, Hoch-

strasser DF. Electrophoresis 18

(1997) 324–327.

Over-swelling of a strip results

in liquid exudation during IEF.

This would cause protein trans-

port to the surface, resulting in

background smearing.

And the separation time will be

longer.

1.5 Two-dimensional Electrophoresis 81

According to a theoretical calculation the volumes for the 3 mm wide

and 0.5 mm thin strips would be 360 mL for a 24 cm and 270 mL for

an 18 cm strip. In practice the optimal reswelling volumes are:

7 cm strip 125 mL

18 cm strip 340 mL

24 cm strip 450 mL

The solutions are pipetted into the grooves as streaks; the strips are

placed on top of the solutions, and are covered with cover fluid (paraf-

fin oil) to prevent crystallization of the urea. Reswelling is performed

at room temperature, because the urea would crystallize in a cold

room. Figure 1.24 shows a reswelling tray for up to 12 strips of differ-

ent strip lengths up to 24 cm.

Fig. 1.24: IPG Dry strip reswelling tray for rehydration of

IPG strips in individual grooves.

Cover fluid The gel strips are covered with paraffin oil during rehy-

dration and IEF to prevent drying of the strips, crystallization of the

urea, and oxygen and carbon dioxide uptake. It has never been

observed that hydrophobic proteins change over from the rehydration

solution into the oil phase.

Rehydration time As mentioned above, there are two ways to apply

the sample to these immobilized pH gradient strips rehydration load-

ing and loading the samples on pre-rehydrated IPG strips.

Rehydration time:

Without sample >6 h

Including sample >12 h or overnight *)

These values are empirical and

have been determined and

optimizes with practical experi-

ments.

Silicon oil is not recommended,

because it can contain dissolved

oxygen and it is not easy to

remove from the instruments

and the benches.

*) The large protein molecules

need a long time to diffuse into

the strip.

1 Electrophoretic Techniques82

1.5.3.2 Sample Application onto IPG Strips

As shown in Figure 1.25, there are several ways to apply the sample

to immobilized pH gradient strips:

Rehydration loading. The extracted or solubilized

sample is diluted with rehydration solution to

the wanted amount of protein. Rehydration of a

strip can be carried out in an individual strip

holder or in a reswelling tray in an individual

groove with a defined volume (see above, rehy-

dration). The dry gel matrix takes up the fluid

together with the proteins. Because the pores are

very small in the beginning, the smaller mole-

cules (water, urea, detergent, and reductant) go

into the gel matrix faster than the proteins.

Many of the proteins diffuse into the fully rehy-

drated gel later. This is the reason for the much

longer rehydration time required for rehydration

loading (see above). IEF can be performed with

the gel surface up or down depending on the

accessory or equipment used. Figure 1.29 shows

the strip holder concept, where active rehydra-

tion (rehydration under low voltage) can be per-

formed, and the voltage steps and gradients for

IEF can automatically applied.

Cup loading. The strip is pre-rehydrated with

rehydration solution. The sample is applied into

a loading cup either on the anodal or the catho-

dal end. Depending on the equipment a maxi-

mum of 100 mL or 150 mL sample can be applied.

The optimal application point is critical, and has

to be determined for each sample type and the

gradient used. The proteins are transported into

the strip electrophoretically. In this case IEF is

always performed with the gel surface up.

Between the electrodes and the IPG strip ends

filter paper pads are applied. The electrode pads

are soaked with distilled water and blotted with a

tissue paper to become damp, not wet.

Paper bridge loading. For application of very high

sample volumes a modification of the cup load-

ing principle has been proposed: paper bridge

loading. Solution containing up to 5 mg protein

can be loaded on an 18 cm long narrow pH

Note: the more diluted the

sample, the easier the proteins

enter the gel.

Sabounchi-Schtt F, Astrçm J,

Olsson I, Eklund A, Grunewald

J, Bjellqvist B. Electrophoresis

21 (2000) 3649–3656.

1.5 Two-dimensional Electrophoresis 83

range IPG strip (Sabounchi-Schtt et al. 2000). A

large sample volume requires that a large paper

pad be applied at the other end of the IPG strip

to absorb excess water. Figure 1.25 D shows the

arrangement used when sample is applied to a

paper bridge positioned between the anode and

an IPG strip. The electrode pads are soaked with

distilled water and blotted with a tissue paper to

become damp, not wet. Sample solution is

applied to the paper bridge (500 mL). With anodic

application the anode is positioned as far out as

possible in the electrode holder, while the cath-

ode is positioned close to the end of the IPG

strip to ensure good contact between electrode

pad and IPG strip.

Fig. 1.25: Schematic representation of the different ways of sample

application on an IPG strip: A. Rehydration loading in a strip holder

for active rehydration and impress the voltage on it for IEF. B. Passive

rehydration loading in a reswelling tray. C. Cup-loading for up to

150 mL sample solution. D. Paper bridge loading for up to 500 mL

sample solution. For C and D anodal application is shown, for some

samples and gradients it might be better to apply the sample at the

cathodal end.

1 Electrophoretic Techniques84

Kane et al. (2006) have reported that for basic pH gradients they get

consistently better results with paper bridge loading than with cup-

loading. Furthermore, the proteins with lower isoelectric points stay

in the paper bridge and can be reloaded on acidic IPG strips – with

application at the cathodal side. In this way it is possible to apply pre-

cious, but limited samples onto several gels with different pH ranges.

& Note: The application point (anodal or

cathodal) is of importance to obtain good

results.

The way of sample loading will have an effect on the 2-D electrophor-

esis pattern. This is easy to understand: Each protein has an individu-

ally shaped titration curve; the mobility of the protein is usually differ-

ent, depending on whether it is negatively or positively charged.

Rehydration loading: Each protein exists in both

ionic forms: negatively and positively charged,

the proteins approach their pI from both sides

with different mobilities.

Cup or paper bridge loading: All proteins are

charged in the same way, all proteins approach

their pIs from the same side.

These facts can explain why some samples show better results with

cup loading compared to rehydration loading.

The following table lists the advantages and disadvantages of the

two methods.

Tab. 1.2: Comparison of rehydration and cup / paper bridge loading.

Rehydration loading Cup loading and paper bridge loading

Pro Pro

The proteins are distributed evenly

over the gradient, precipitation at

the sample application point cannot

happen.

IEF in very acidic narrow gradients,

in basic pH gradients, and HED

containing strips work much better.

Rehydration, sample application and

IEF can be combined to one single

step, resulting in less manipulation

steps.

The sample gets faster transported into

the gel and becomes separated, thus the

chances of protein interactions are

reduced.

When the sample is diluted, a higher

sample volume can be applied.

If necessary, the proteins can be loaded

onto both ends of the strip in order to

include proteins with pIs close to the

pH of their application points.

Voltage can be applied during

rehydration, which improves the

entry of high molecular weight

proteins.

For some sample types it is beneficial,

when all proteins are charged in the

same way, the molecules repel each

other and do not form aggregates.

Kane LA, Yung CK, Agnetti G,

Neverova I, Van Eyk JE. Proteo-

mics 6 (2006) 5683–5687.

See page 40: titration curves.

See Table 1,2 below, under

“Pro, cup loading”

1.5 Two-dimensional Electrophoresis 85

Rehydration loading Cup loading and paper bridge loading

Con Con

When basic gradients like pH 6–9,

pH 6–11, or pH 7–11 are employed,

rehydration loading leads to severe

protein losses, because several

proteins aggregate in basic environ-

ment.

Proteins with isoelectric points close to

the pH of the application point have low

mobility and solubility, they tend to

precipitate on the surface and build a

vertical streak in the second-dimension

gel.

If DeStreak is added to the rehydra-

tion solution, the final concentration

of DTT must not be higher than

1 mmol/L the use of reductant is

thus limited.

The procedure is more laborious and

takes longer time than rehydration

The protein mixture is kept for many

hours at room temperature, thus the

danger of exposure to protease

activities can increase.

Also when proteins are applied on both

ends, proteins precipitate at the appli-

cation points.

During the rehydration process the

protein concentration outside the gel

strip increases, which can lead to

aggregation of proteins; those cannot

enter the gel. Often they become

visible in the 2

dimension as hori-

zontal streaks.

During the first phase of IEF the

proteins become concentrated at the

point of entry and can form aggregates

and precipitate.

In several papers the statement can be found that higher protein

loads can be applied with rehydration loading than with cup loading.

This should be checked for each sample type, for some samples it

might be vice versa.

A strategy for selection of the optimal procedure is described later.

1.5.3.3 IEF Conditions

In general, separation conditions have to be adjusted to the nature

and composition of the sample. Horizontal streaking in the 2-D pat-

tern, for instance, can have many different reasons: overloading

effects, too short focusing time, too long focusing time (some pro-

teins became unstable at their pIs), oxidation of cysteines, too much

salt, nucleic acids, phospholipids etc.

Gel surface up or down Running IPG strips with the gel surface up

– as shown in Figure 1.25B, C, D – has the advantage of employing

filter pads at the gel ends, which take up salt ions and proteins with

pIs lying outside the pH gradient of the IPG strip. Another benefit is

the open surface just covered with paraffin oil: when highly abundant

Gels with high protein loads

should always be run with the

surface up, also when rehydra-

tion loading is performed, see

page 95.

See page 97.

For some sample types optimi-

zation by a series of pre-experi-

ments can be necessary.

This can reduce horizontal

streaking considerably.

1 Electrophoretic Techniques86

proteins form ridges, there will not be any mechanical pressure on

them (see Figure 1.32).

Temperature It is very important to run IEF at a defined tempera-

ture, even when the proteins are denatured. It had been demon-

strated that spot positions of certain proteins can vary along the x axis

of the gel dependent on the temperature (Gçrg et al. 1991). Running

the strips at 20 C is optimal, because it is above the temperature

where crystallization of urea can be critical, and it is below overheat-

ing temperature, which can cause carbamylation of proteins during

IEF. Even, when the applied electric power, the product of current

and voltage, is very low in IPG strips, it is not sufficient to run IEF

just in a thermostated room with 20 C. Active temperature control is

necessary to dissipate local heat developed at the ion fronts in the

strips (“hot spots”, see Figure 1.26). Aluminum oxide ceramics has

the best thermal conductivity, and affords far the best temperature

dissipation. It is therefore the preferable material for strip holders

and manifold accessories.

Fig. 1.26: Schematic representation of the distribution of salt and

buffer ions in an IPG strip during IEF and the resulting electric field

strength. The ion fronts are migrating slowly towards the electrodes.

V = voltage, E = electric field strength, d = electrode distance.

Hot spots: In an IPG strip, there are always remains of salt and buf-

fer ions present, which have not been washed out. They are bound to

the buffering groups of the immobilized pH gradient. Additionally

there are ions, which originate from ionic sample contaminants.

In the electric field they start to migrate towards the electrodes with

the opposite signs. During the run they are forming visible ion fronts

between the region with low ion concentration and the regions with

high ion concentrations (Figure 1.26), which are moving towards the

electrodes. Immobilized pH gradients have very low conductivity,

because the buffering groups of the gradient are fixed and cannot dif-

Gçrg A, Postel W, Friedrich C,

Kuick R, Strahler JR, Hanash

SM. Electrophoresis 12 (1991)

653–658.

Electric conditions and

temperature control are closely

related.

In IEF, all non-amphoteric

compounds are transported out

of the gradient towards the

electrodes.

1.5 Two-dimensional Electrophoresis 87

fuse. Because of the differences in conductivity, the electric field

strength in these regions is varying considerably. Punctual heat devel-

opment can be observed at these fronts, so called “hot spots”.

Electric conditions As already mentioned above, immobilized pH

gradients have very low conductivity. The current is usually limited at

50 to 70 mA per strip. Higher current settings are not recommended,

because the strips could start to overheat and burn in certain areas of

the gradient. The strips should never be prefocused, because the

higher conductivity produced by salt and buffer ions in the beginning

is advantageous for sample entry and for the start conditions of pro-

teins.

The electric conditions are controlled with the voltage setting. First

low voltage is applied in order to avoid sample aggregation and preci-

pitation in the loading cup or overheating in some strip areas. The

voltage is slowly raised to reach electric field strength as high as pos-

sible in the focusing phase.

& Note: The set current can limit the achievable

voltage, when the sample contains too much

salt, or when buffers are included in the

rehydration or lysis solution.

The voltage changes are either programmed in steps or with ramping,

applying voltage gradients.

Often the instructions supplied with ready-made IPG strips suggest

relatively high voltage start conditions, like 500 V for an hour. These

settings can work well for various samples like rat liver extract or E.

coli lysate, but there are a number of samples which require lower

and slower starting conditions. Particularly samples with high con-

tents of high molecular weight and hydrophobic proteins require a

low electric field for a relatively long time in the beginning. This is

true for all ways of sample application: rehydration, cup, and paper

bridge loading.

When a new sample type is separated for the first time, it is highly

recommended to apply the safest settings. In Figure 1.30 and in the

practical part of this book (see page 314 ff) low voltage settings over

longer time are suggested. They represent the “worst case” for

unknown samples based on experiences by Burghardt Scheibe.

The electric conditions change sometimes dramatically during the

first phase as shown in Figure 1.27:

Burghardt Scheibe: personal

communication.

1 Electrophoretic Techniques88

Fig. 1.27: Development of the actual voltage course during the

first IEF phase in an IPG strip.

The separation can be speed up with applying higher electric field

strength. For these high voltages the instruments have to be designed

to comply with a high safety level.

Volt hours The complete voltage load is defined in volt hour inte-

grals (Vh): the amount of voltage applied over a certain time. For

example: 5,000 Vh (5 kVh) can be 5,000 V in one hour or 1,000 V over

five hours.

The Vh definition corrects the running condi-

tions for different conductivities in different

strips caused by different protein compositions

and salt loads.

In many cases increasing the voltage gradients

(ramping) rather than in steps improves the

result considerably. In order to apply comparable

voltage loads, the Vh value is a good measure-

ment.

Underfocusing When the applied volt hours are insufficient, not all

spots are round. Horizontal streaks are produced instead. Higher volt

hours loads are needed for samples containing a higher number of

high molecular weight proteins, more hydrophobic proteins and for

preparative runs.

Overfocusing When the proteins are focused too long, some other

negative effects can happen:

Cysteines become oxidized, the pI of the protein

changes.

Some proteins become instable at their isoelec-

tric point. The modified proteins or its frag-

ments have different pI and start to migrate

These effects are result of too

long time, not of too high

voltage.

1.5 Two-dimensional Electrophoresis 89

again. The result: some horizontal streaks com-

ing out from a few spots.

Basic narrow gradients are particularly sensitive

to over focusing. Additionally to possible protein

breakdowns, the basic extreme (> pH 10) of the

gel can become instable because of the very high

pH.

& For basic pH gradients the best results are

obtained with a focusing phase as short as

possible at a voltage as high as possible.

Should the optimal Volt hours have been achieved during the night

or another time of no attendance, the separation will stop after the

programmed value has been reached. Instead of applying a low

“cruising voltage” no voltage should be applied on the strip. Prior to

removal the strips from the strip holders the highest possible voltage

is applied for 15 minutes, in order to refocus the slightly diffused

bands.

Sample entry effects During the first phase of IEF with the cup load-

ing procedure complex phenomena are observed:

If the voltage setting in the beginning is low

(100 V, 18 cm IPG strip), the proteins enter the

gel, but a part of them precipitates or gets stuck

within the first few cm of the strip, when the vol-

tage is raised too quickly after this phase. As a

result of this a vertical streak can be found in the

second-dimension gel, which is displaced a few

centimeters from the application point.

If the voltage setting for the start is high (like

500 V on a 18 cm IPG strip), a part of the pro-

teins accumulate and precipitate on the surface

of the application point and show a vertical

streak in the second dimension. Sometimes the

precipitated proteins at the application point are

washed off during equilibration: and then no

streak is visible.

& These matters are critical: the voltage level during

sample entrance and the beginning of protein

migration, and the duration of the focusing phase

at high voltage.

Some manuals suggest

applying a low “cruising”

voltage of 500 V to maintain

the band sharpness. This is not

the optimal procedure.

Josef Blles, personal commu-

nication.

It should be continued to use

low voltage during the first few

hours.

In the second case the 2-D

separation shows a clearer

pattern than in the first.