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

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1 Electrophoretic Techniques100

1.5.4

Second Dimension: SDS Electrophoresis

1.5.4.1 Gel Types

Homogeneous gels

For 2-D separations mostly a homogeneous gel

with 12.5% T and 3% C is used; the proteins of major interest in the

size range from 10 to 100 kDa are optimally resolved. For special

cases a table with optimal gel concentrations for different protein size

ranges can be found in the GE Healthcare 2-D Handbook (2005).

However, as demonstrated by Langen et al. (1997), it is not easy to

predict the optimal acrylamide concentration according to a mathe-

matical function: In an example shown in this paper a group of pro-

teins with ca. 50 kDa, which were co-migrating in 9–16% T gradient

gels were best resolved in a gel with 7.5% T. This shows that a try and

error procedure must be applied for optimization, when certain pro-

tein groups need to be completely resolved.

Gradient gels Generally, in gradient gels the overall separation inter-

val is wider; also the linear relation interval between the logarithm of

and the migration distance is wider than for homogeneous gels.

Also the spots are sharper because the pore sizes are continuously

decreasing. This effect can be very useful for separating proteins,

which are highly glycosylated. However, when homogeneous gels are

run under optimal conditions, resolution and spot definition is high

enough: a gradient gel is not necessary.

Gradient gels have the following shortcomings:

The preparation of gradient gels is much more

work.

It is more difficult to obtain reproducible gel

properties than for homogeneous gels.

The gels swell unevenly during staining, and

show uneven background intensity.

Horizontal flatbed gels Because the IPG strip is just placed on the

surface of the horizontal gel, a stacking gel with low acrylamide con-

centration is necessary for optimal protein transfer and separation

(Gyenes and Gyenes, 1987). Resolving and stacking gel is usually

polymerized in one piece. These gels can only be cooled from one

side; rarely gels thicker than 0.5 mm are used. These gels are poly-

merized onto a support film.

Laboratory-made gels are usually run with liquid buffers, con-

nected to the gel with paper wicks. The electrodes are then placed

into the buffer tanks. The Laemmli buffers are used. Instructions and

GE Healthcare Handbook. GE

Healthcare Life Sciences 80-

6429-60 (2005).

Langen H, Rçder D, Juranville

J-F, Fountoulakis M. Electro-

phoresis 18 (1997) 2085–2090.

Gyenes T, Gyenes E. Anal

Biochem 165 (1987) 155–160.

Gçrg A, Weiss W. In Rabilloud

T, Ed. Proteome research: Two-

dimensional gel electrophoresis

and identification methods.

Springer Berlin Heidelberg New

York (2000) 107–126.

1.5 Two-dimensional Electrophoresis 101

recipes for preparing gels for flatbed systems in the laboratory can be

found in several books: Gçrg et al. (2000) and Westermeier (2004).

Vertical gels For 2-D electrophoresis in vertical systems the stacking

gel is not needed, because:

The proteins are already pre-separated by IEF

and will therefore not aggregate while they enter

the resolving gel.

The proteins migrate from a gel into another gel,

and not from a liquid phase into a gel.

Still a discontinuous buffer system is employed: the gel buffer com-

position is different from the running buffer. The spots are well

resolved, because there is a stacking effect happening between the

highly mobile chloride in the gel and the lowly mobile glycine in the

running buffer.

Omitting the stacking gel solves a few technical problems and

makes the procedure easier:

The edge between the stacking and resolving

gels contains incompletely polymerized acryla-

mide monomers and oligomers, which stick to

proteins and partly modify proteins.

Some proteins get caught between the stacking

and resolving gels.

One of the problems sources with reproducibil-

ity is abolished.

Ready-made gels cannot contain a stacking gel

with a buffer different from the resolving gel

because of diffusion.

The additional step of polymerizing a stacking

gel short before the run is abolished.

The standard thicknesses of SDS slab gels run in vertical equipment

are 1.0 mm and 1.5 mm. Thinner gels cannot be used, because the

IPG strip including the film support is 0.7 mm thick, and it swells

during equilibration with SDS buffer.

Tab. 1.4: Comparison of gel thicknesses.

Advantages of 1.0 mm gels: Advantages of 1.5 mm gels:

Faster separations possible, leading to sharper

spots

Higher loading capacity

Detection methods faster and more sensitive Higher mechanical stability

Protein / gel volume ratio more favorable for

tryptic digestions resulting in higher peptide yield

Westermeier R. Electrophoresis

in Practice. WILEY-VCH, Wein-

heim (2004).

Therefore the upper running

buffer must not contain any

chloride ions.

The IPG strip is placed onto the

SDS gel sideways.

1 Electrophoretic Techniques102

1.5.4.2 Gel Casting

First of all, it is very important to clean glass plates and equipment

very thoroughly. Mass spectrometry analysis is very sensitive, and

every contamination will show up in the mass spectrogram and

might lead to wrong results. Keratin stemming from hair and skin is

the most frequently found protein contamination; sometimes even in

reagents purchased from a supplier. It has become general practice in

most proteomics laboratories to filter each monomer and buffer solu-

tion through a membrane filter before use.

Also for the SDS PAGE step only high-quality chemicals should be

used. Particularly for such a sophisticated and multistep procedure

like 2-D electrophoresis the search for trouble causing sources is very

cumbersome and time-consuming.

When the sample proteins have been pre-labeled with fluorescent

dyes, the gels are scanned inside the cassette. Therefore only low-

fluorescent glass is used for DIGE experiments.

For accurate spot picking the gel have to be fixed to one of the glass

plates, the glass plate without the spacers is therefore treated with

Bind-Silane prior to assembling the cassettes. If the Bind-Silane treat-

ment is performed long time before the cassettes are assembled, non-

treated and treated glass plates should be kept separate. It has been

observed that Bind-Silane molecules diffuse towards the non-treated

glass surface and cause strong adherence of the gel to the second

glass plate.

& Meticulously cleaned instruments and glass plates

as well as high-quality reagents are of high

importance.

Gels for miniformat and medium size instruments can be cast as sin-

gle gels (see Figure 1.35) as well as in multicasters, which are built

similar to those used for large gels (see Figure 1.37).

Large format gels, which are usually used in proteomics, can only

be prepared in multicasters, because the glass plates of the cassettes

would bend out and form a bulbous gel with a higher thickness in

the center than along the edges.

For the handling of multiple gels it is not very practical to use glass

plates with separate spacers and clamps. Therefore the cassettes con-

tain fixed spacers and a hinge on one side, as shown in Figure 1.36.

However, when the gel is to be fixed to one of the glass plates with

Bind-Silane for spot picking, non-hinged cassettes are used, with the

spacers attached to the second glass plates.

The author of this chapter

could write a detective story on

pitfalls caused by dirty or old

reagents.

For a high throughput method

anyhow multiple gels are

needed.

For multiple gels it is practical

to insert a little piece of paper

with a key number for easy

identification of the sample.

1.5 Two-dimensional Electrophoresis 103

Fig. 1.35: Casting single miniformat and medium size SDS

polyacrylamide gels for 2-D electrophoresis

Fig. 1.36: Glass cassette for multiple SDS gel casters

and instruments. Spacers are fixed; glass plates are

connected with a hinge.

Depending on how many gels to be cast at a time, different types of

multiple gel casters have been designed. During pouring the mono-

mer solution into the caster the liquid will not only flow between the

spacers, but also between the cassettes. For easy separation after the

polymerization plastic sheets have to be inserted between the cas-

settes. Originally they are designed to introduce the monomer solu-

tion into the cassettes from the bottom. This is necessary when gradi-

ent gels are prepared.

As already mentioned, the polymerization process is an exothermic

reaction. When multiple-gel casters are used, the composition of cata-

lysts in the polymerization solution is more critical than for single gel

Without the plastic sheets, the

cassettes would stick together

because of the polymerized gel

layers.

This practical hint is mentioned

here, because some instructions

incorrectly suggest the opposite

procedure.

1 Electrophoretic Techniques104

casting. If the polymerization runs too fast, very much heat is pro-

duced in the center of the multicasters, leading to thermal convec-

tion, which results in curved gel edges and irregular polymerization.

It is very practical to add the TEMED to the other constituents already

when the monomer solution is mixed together. It will not cause any

start of polymerization. The ammonium persulfate, however, must

not be added before pouring the gel.

It must be avoided that the start of polymerization begins while the

monomer solution is still flowing. Furthermore the multiple solu-

tions in the cassettes need a certain time to level out following the

principle of the communicating vessels. The monomer solution

should be degassed with a vacuum pump and then precooled in a

refrigerator at 4–8 C. This measure reduces the heat development

and delays the start of the polymerization. It has several advantages:

The solution has time enough to level out

between the cassettes and settle until the solu-

tion starts to polymerize: this prevents irregular

gel composition and variation in gel lengths.

Local overheating is avoided, which can result in

local variations of sieving properties of the gels.

Gels properties are more reproducible indepen-

dent of the seasonal temperatures in the labora-

tory.

When the room temperature is higher than 20 C, the multicaster

should – like the monomer solution(s) – be pre-cooled in a cold room

or a refrigerator.

For homogeneous gels it is quicker to fill the caster from the top by

pouring the solution directly into the cassettes (see Figure 1.37A).

Before the polymerization starts, 0.1% (w/v) SDS–water solution is

sprayed into the cassettes with a finger-pumped plant sprayer to

achieve straight upper gel edges (see Figure 1.37B). The very small

detergent solution droplets glide down on the glass surface very

quickly and will not mix with the monomer solution, because they

are very small and have a low density. This procedure gives much

straighter edges than pipetting water-saturated butanol or 70% (v/v)

isopropanol into the cassettes, as it is proposed in most instructions.

Pipetting produces larger drops, alcoholic solutions have a high affi-

nity to the vinyl spacer material and can cause bent surfaces.

& Note: It is very important to obtain a straight

gel surface, because the IPG strip is placed on

the gel edge-to-edge: in this direction the film

support is absolutely rigid and cannot be bent to

follow a curved edge.

Most of the stock solutions are

anyhow kept in the refrigerator.

When alcoholic overlay solu-

tions are used, they need to be

removed an hour later, because

they gnaw at the plastic surface

of the caster box. SDS solution

does not need to be removed.

1.5 Two-dimensional Electrophoresis 105

Fig. 1.37: Casting multiple homogeneous SDS gels.

A. Pouring the monomer solution directly into the cassettes.

B. Overlaying the edges by spraying 0.1% (w/v) SDS–water

solution into the cassettes.

One should not forget that the quality and the age of chemicals, parti-

cularly the catalysts, greatly influence the polymerization efficiency of

the monomer solution. For instance: The TEMED can be stable for

several years, but it can suddenly decay within a week. Therefore it is

advisable to replace the TEMED every year with a new bottle.

Gradient gels For the preparation of gradient gels a gradient maker

has to be connected to the gel caster. Here there is no other way than

casting the gels from the bottom. The gradient solution has to be

delivered to the box slowly to avoid mixing – taking 5–10 minutes.

The speed can be adjusted by using a laboratory peristaltic pump, or

by selecting a certain level for the liquid beaker and reducing the dia-

meter of the tubing with a pinchcock clamp (see Figures 4.4 and 4.6

on pages 334 and 341).

Gels for spot picking After the 2-D patterns have been compared

with image analysis software, proteins of interest have to be identified

and characterized. For these further analyses the gel plugs containing

these proteins have to be cut out of the gel slab. For high accuracy,

reliability, and automation of this procedure robotic spot pickers are

employed (see pages 138 and 140 f). The most accurate and reliable

way is to use the x/y coordinates of the respective spots from the

image analysis results in the automatic spot cutter. It is, however,

important that the gel does not change its shape by shrinking or swel-

ling. Therefore gel slabs have to be fixed to a rigid film or glass plate

backing.

Note: The quality of the edge is

influenced by the quality of the

chemicals.

Using a pump means a lot of

work. Everything has to be set

up very quickly, because the

casting and the overlaying

procedure must be finished

before the gel solution starts to

polymerize. The casting proce-

dure must be as exact as

possible to prepare reproducible

gels.

1 Electrophoretic Techniques106

Gels fixed to a glass plate Because it is not easy to bind a gel to a

glass plate after staining, gels are covalently bound to a film support

or glass plate during polymerization. Laboratory made gels are

usually polymerized onto a glass plate, which has been treated with

Bind-Silane. Because staining solutions can diffuse into the gel only

from one side, the gel should be as thin as possible for fast and effi-

cient staining. These gels are usually 1.0 mm thick. Fluorescence

labeled or fluorescence stained gels must be attached to non-fluores-

cent glass. Because this type of glass is very expensive, the gel layer is

removed from the glass plate after all analysis has been done. When

the glass plates are reused, the gel layer is removed with a plastic scra-

per. Remaining gel pieces disappear after vigorous treatment with a

dishwashing brush.

Meanwhile a non-fluorescent support film material has been devel-

oped, which allows using ready-made gels for the combination fluor-

escent detection – spot picking.

Ready-made gels for vertical systems Ready-made gels are more

expensive than laboratory-cast gels. However, one should not forget

that gel casting is a lot of work – particularly for cleaning the equip-

ment – and working time costs money as well. Commercially pro-

duced gels are prepared according to GMP industry standards, and

they are quality controlled.

There are two different concepts:

Gels in glass or plastic cassettes. The handling does

not differ from that of laboratory-cast gels. The

glass cassettes are either sent back to the produ-

cer or they are disposed of.

Gels on film-support. The 1 mm thick gels are

inserted into specially designed re-usable cas-

settes (Figure 1.38). Conventional glass cassettes,

as shown in Figure 1.36, cannot be used,

because air pockets between the glass plate and

the backing develop mechanical pressure on the

gel, leading to an irregular front.

These 12.5% homogeneous polyacrylamide gels contain PPA-chloride

pH 7.0 instead of the standard Tris-chloride buffer pH 8.8, in order to

achieve a long shelf life. Because polymerization at pH 7.0 is more

efficient than at pH 8.8, the gels have a very high mechanical stability,

and the patterns are highly reproducible. Bis-Tris buffer containing

gels are a practical alternative (see above). The gels are packed airtight

in flexible aluminum bags.

It is not easy to produce these

vertical gels on a film support in

the laboratory, because the

polyester films are flexible. To

set up a casting system for film-

supported gels would mean a

big investment in equipment.

It is very annoying, when the

valuable sample is not sepa-

rated well, just because of a

little mistake occurring while

preparing gels.

The sieving properties of a

12.5% T PPA gel are compar-

able to those of 14% T poly-

merized with a Laemmli buffer.

1.5 Two-dimensional Electrophoresis 107

Fig. 1.38: Support cassette and ready-made film-backed

vertical gel. The gel is shorter at the upper side to

accommodate the IPG strip.

Kits with buffer concentrates and agarose sealing solutions are

available. The kit for the film-backed PPA gels contains also a bottle

with the gel buffer. The best way to insert the gel into the cassette is

to apply a streak of 1 mL of the gel buffer onto the glass plate along

the spacer of the closing side, and placing the gel on the glass plate

first touching the buffer streak with the respective edge. The gel posi-

tion can be easily adjusted that the edge at this side is in contact along

the entire spacer and the lower gel edge is flush with the edge of the

glass plate. When the gel is lowered on the rest of the glass plate care-

fully, no air bubbles are caught between gel surface and glass plate.

The liquid near the closing side and some air bubbles – mostly close

to the hinged side – are squeezed out with a roller.

A narrow gap of less than 1 mm remains between one spacer and

the gel edge: this will be sealed with hot agarose solution after the

IPG strip has been inserted.

For Western blotting analysis or for high sensitivity fluorescent

staining the support film has to be removed. It can be cut off with a

simple instrument using a steel wire (see Figure 1.39). The large for-

mat gels are easy to handle also without the film-backing because

they are well polymerized.

This procedure works only with flexible support films, not with

rigid, Bind-Silane treated glass plates.

Of course, also these cassettes

have to be thoroughly cleaned

before and after each use to

avoid contaminations.

A liquid layer between gel

surface and glass plate has to

be avoided. It would cause

blurred spots.

Note: For those who have only

used film-backed gels, handling

of non-backed slab gels requires

some practice and skill.

1 Electrophoretic Techniques108

Fig. 1.39: Film remover for complete removal of a flexible

film-backing from the gel.

1.5.4.3 Instrument and Gel Setup for the Second Dimension

Figure 1.40 shows a Multiphor flatbed chamber used as SDS PAGE

instrument and a mini vertical electrophoresis system for miniformat

gels. These two instrument types are not for high throughput and

thus mostly employed for optimization work.

Fig. 1.40: Flatbed and vertical mini electrophoresis apparatus for SDS PAGE.

In the flatbed apparatus the standard gel size is 2519 cm. The elec-

trode distances can be adjusted to shorter separation distances. For

example, SDS electrophoresis of three miniformat IPG strips with

7 cm length can be run together in one gel under identical conditions

(see Figure 1.41).

1.5 Two-dimensional Electrophoresis 109

Fig. 1.41: Three miniformat 2-D separations run in one flatbed gel.

IEF in three 7 cm IPG strips 4 to 7, SDS PAGE in a 2511 cm gel with

a stacking zone of 5% T and a homogeneous resolving area of

12.5% T, 0.5 mm thick. Silver staining.

Ready-made gels for vertical miniformat chambers are available from

several suppliers. An alternative is to run also several short IPG strips

together on a larger vertical gel. Some vertical systems can also be

equipped with shorter glass plates.

Tab. 1.5: Comparison of the properties of flatbed and vertical

systems.

Flatbed Systems Vertical Systems

One gel per instrument. Multiple gels runs are possible.

Can also be used for IEF in IPGs. Dedicated for SDS electrophoresis.

Gel thickness is limited, because cooling

is only possible from one side.

Higher protein loading capacity,

because thicker gels can be used,

which are cooled from both sides.

Very versatile for different gel sizes

and methods.

Gel sizes fixed by glass plate sizes.

Thin layer gels can be used, easy

application of the IPG strips.

Thin gels cannot be used, because

the IPG strip would not fit between

the glass plates.

Buffer strips (polyacrylamide or filter

paper) can be used instead of large

volumes of liquid buffers.

Blotting is easier because of higher

gel thickness.

The IPG strip has to be removed from

the SDS gel after 40 minutes because

of EEO effects.

The IPG strip does not have to be

removed.

Gçrg et al. (1995) have performed a systematic comparison of

results, obtained with a flatbed and a vertical system for the same

Up to 3 mm thick gels can be

used in vertical systems

Very thin gels show higher sensi-

tivity of detection, they are

easier and quicker to stain.

With flatbed systems there is

reduced chemical and radioac-

tive liquid waste.

EEO occurs in both systems,

therefore urea, glycerol in the

equilibration buffer and low

initial voltages are needed.

Gçrg A, Boguth G, Obermaier

C, Posch A, Weiss W. Electro-

phoresis 16 (1995) 1079–1086.