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

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1 Electrophoretic Techniques20

The ionic strength should be as low as possible to keep Joule heat

development to a minimum. And, the higher the ionic strength of

the buffer, the lower will be the mobilities of the sample ions.

In isoelectric focusing a different buffer concept is applied: either

amphoteric buffering components migrate to an endpoint in the sys-

tem and become decharged, or the buffering groups are fixed to the

static separation medium.

Another special case is free flow electrophoresis (FFE).

1.1.1

Free Flow Electrophoretic Methods

In the free-flow approach, originally developed by Hannig (1982), a

continuous stream of buffer flows in a 0.5–1.0 mm wide layer inside

a cooled glass cuvette. The buffer is supplied to the cuvette over the

entire width. At one end the sample is injected at a defined spot, and

at the other end the fractions are collected in an array of tubes. The

electrical field is applied perpendicular to the buffer flow.

The varying electrophoretic mobilities perpendicular to the flow

lead to differently strong but constant deviations of the components,

so that they reach the end of the separation chamber at different

though stable positions (see Figure 1.1).

Fig. 1.1: Schematic drawing of

continuous free flow electrophor-

esis, according to Wagner et al.

(1989).

Isoelectric focusing will be

described later in detail.

In contrast to all other methods

FFE is a continuous system.

Hannig K. Electrophoresis. 3

(1982) 235–243.

Wagner H, Kuhn R, Hofstetter

S. In: Wagner H, Blasius E. Ed.

Praxis der elektrophoretischen

Trennmethoden. Springer-

Verlag, Heidelberg (1989)

223–261.

1.1 The Principle of Electrophoresis and Some Methodological Background 21

Different separation methods can be applied: zone electrophoresis in

homogeneous and discontinuous buffer systems, and isoelectric

focusing (see below).

Discontinuous field electrophoresis is a specialty of free flow electro-

phoresis with a zone concentration effect: The sample solution is sup-

plied in a wide zone through the central openings, the buffer solu-

tions on the right and on the left hand side have a twenty times

higher conductivity than the sample solution. The sample ions are

more or less strongly deflected towards the anode or the cathode,

depending on their charges. When they reach the borderline between

sample and buffer stream, their electrophoretic mobility is consider-

ably reduced, resulting in a concentration of the fraction at the bor-

derline.

Free flow electrophoresis is the method of choice for separating

large particles like sub cellular components, as shown in the paper by

Zischka et al. (2003).

1.1.2

Gels for Electrophoretic Techniques

Most electrophoretic separations in proteomics are performed in gel

matrices. There are basically two types of gels: granulated and com-

pact gels. Granulated gels, like dextranes, are almost exclusively used

for chromatography, compact gels for electrophoretic techniques.

There is just one exception: dextrane gels are well suited for prepara-

tive applications and pre-fractionation according to charge, as shown

below. A detailed description for working with dextrane gels for pre-

parative IEF can be found elsewhere (Westermeier, 2004).

The authors are not aware of any proteomics applications in agar-

ose gels; therefore we describe only polyacrylamide gels.

1.1.3

Electroendosmosis Effects

This phenomenon occurs, when fixed charges belonging to the gel

matrix or to glass surfaces are present in an electric field. Those are

for instance carboxylic groups in gels and silicium oxide groups on

the glass surface. These groups become ionized in basic and neutral

buffers: in the electric field they will be attracted by the anode. As

they are fixed in the matrix, they cannot migrate. In order to maintain

the physico-chemical balance, this will be compensated by the coun-

terflow of H

ions towards the cathode. This effect is called electro-

endosmosis. In practice, this phenomenon is observed as a water

flow towards the cathode, which carries proteins along with it and

causes blurred zones and streaking. As described further below, it

All these separations are

performed in a continuous flow.

This one of the rare methods

where a high buffer concentra-

tion is employed for trapping

sample components in a

certain zone.

Zischka H, Weber G, Weber

PJA, Posch A, Braun RJ,

Bhringer D, Schneider U,

Nissum M, Meitinger T,

Ueffing M, Eckerskorn C.

Proteomics 3 (2003) 906–916.

Westermeier R: In Cutler P. Ed.

Protein Purification Protocols.

Second edition. Methods in

Molecular Biology, Volume

244, Humana Press, Totowa,

NJ (2004) 225–232.

1 Electrophoretic Techniques22

can also cause difficulties during the transfer of proteins between

IPG strips and the second-dimension gel.

1.2

Polyacrylamide Gel Electrophoresis

1.2.1

The Polyacrylamide Gel

Polyacrylamide gels are polymerized from acrylamide monomers and

a cross-linking reagent – usually N,N'-methylenebisacrylamide. The

reaction is started with ammonium persulfate as catalyst; TEMED

provides the tertiary amino groups to release the radicals. The pore

size can be exactly and reproducibly controlled by the total acrylamide

concentration T and the degree of cross-linking C:

T ¼

ða þ bÞ · 100

½%; C ¼

b · 100

a þ b

½%:

a is the mass of acrylamide (g),

b the mass of methylenebisacrylamide (g),

V the volume (mL).

When C remains constant and T increases, the pore size decreases.

When T remains constant and C increases, the pore size follows a

parabolic function: at high and low values of C the pores are large.

For T = 10% the minimum lies at C = 4%; with higher C values the

gels become are brittle and relatively hydrophobic. For T = 16% the

minimum lies at C = 6%; these gels are used for separation of small

peptides

For protein and peptide separations T values between 4% and 16%

T are used. It has been observed that higher T values can cause pro-

tein degradation. In practice the applied C values are 2.5–3.0% for

zone electrophoresis and 3% for isoelectric focusing.

Polyacrylamide gels must be polymerized in closed cassettes to

exclude oxygen, which would interrupt the polymer chain formation.

Often the monomer solutions are degassed with the help of a vacuum

pump for optimal polymerization effectiveness. The polymerization

efficiency is additionally influenced by the monomer concentration,

the quality of the reagents, temperature, and pH value. The polymer-

ization is exothermic. Particularly, when a highly concentrated gel

solution polymerizes, a substantial amount of heat is generated. The

higher the temperature, the faster and more efficient is the polymeri-

zation.

When the monomer concentra-

tion is very low and the pH

value very high, the gel will not

polymerize. For instance, it

would be very difficult to poly-

merize a 4%T gel with a pH of

8.8.

1.2 Polyacrylamide Gel Electrophoresis 23

The standard catalyst system with TEMED and ammonium persul-

fate works only for gels containing neutral and basic buffers. Acidic

gels are polymerized with alternative reagents, like ascorbic acid and

ferrous sulfate together with hydrogen peroxide. Preparation of acidic

gels is not trivial, because polymerization occurs very quickly and is

difficult to control.

1.2.1.1 Silent Polymerization

It should be noted that after the crude gel has formed, there is a silent

polymerization following, which takes several hours and completes

the formation of the final matrix. Therefore the gels should not be

used immediately after preparation, and they should not be placed

into a refrigerator or a cold room. The gels need to be left at room

temperature for a couple of hours or overnight. After this they can be

used or stored in the cold. Gels containing an alkaline buffer have a

limited shelf life, because at high pH values the matrix begins to

hydrolyze after a few weeks.

& Practical tip: In order to get reproducible gels,

the concentrations of the catalysts have to be

balanced in favor of a higher TEMED and a

lower ammonium persulfate concentration. At

basic pH, ammonium persulfate can react with

the Tris; this effect is minimized by adding more

TEMED, and by reducing the ammonium

persulfate content.

1.2.1.2 Vertical and Horizontal Gels

In a classic setup polyacrylamide gels are run in vertical chambers

while they are still in the casting cassettes. The samples are applied

into wells formed with a comb during polymerization (see Figure

1.2). As an alternative, the gels can be run with an open surface on a

horizontal flatbed system. Here the sample wells are positioned

within the stacking gel area (see Figure 1.4).

Laboratory-made horizontal gels are usually run with liquid buf-

fers, connected to the gel with paper wicks. The electrodes are then

placed into the buffer tanks. The Laemmli buffers are used. Ready-

made horizontal gels contain a different buffer (see below). Horizon-

tal gels can also be run without the buffer tanks: either thick filter

cardboard, soaked with a concentrated buffer, or ready-made poly-

acrylamide buffer strips, which contain the necessary buffer ions for

one run, are placed on the edges of the gel (see also page 108). In this

case electrode wires are placed on top of these electrode strips. The

If a gel is not perfectly polymer-

ized it will become particularly

obvious during mass spectro-

metry analysis of tryptic digests

of cut-out protein bands or

spots. Incompletely polymer-

ized gels cause high back-

ground in the mass

spectrogram.

A comprehensive guide how to

prepare and run horizontal

polyacrylamide gels can be

found in “Electrophoresis in

Practice” (WILEY VCH).

Kleine B, Lçffler G, Kaufmann

H, Scheipers P, Schickle HP,

Westermeier R, Bessler WG.

Electrophoresis 13 (1992) 73–

75.

1 Electrophoretic Techniques24

buffer strip concept reduces chemical and radioactive liquid waste

considerably (Kleine et al. 1992).

1.2.1.3 Discontinuous Gels

In order to prevent aggregations of proteins or other sample mole-

cules during entering the gel matrix, usually a stacking gel is poly-

merized on top of the resolving gel. The stacking gel contains a differ-

ent buffer composition and has larger pore sizes than the resolving

gel. Figure 1.2 shows how such discontinuous gels are prepared. The

stacking gel is poured about one hour before electrophoresis.

Fig. 1.2: Preparation of a discontinuous polyacrylamide gel. The resolving

gel is polymerized for at least 5 hours or overnight at room temperature.

The stacking gel is poured about one hour before electrophoresis.

Stacking gels are used for one-dimensional separation, when the

applied sample molecules are in liquid phase, like in 1-D SDS electro-

phoresis, cationic detergent acidic electrophoresis, and Blue native

electrophoresis.

1.2.1.4 Gradient Gels

Porosity gradients are prepared by continuously changing the acryla-

mide concentration in the polymerization solution while pouring the

gel, so that the concentration in the casting cassette decreases from

bottom to top. The density of the highly concentrated solution is sup-

plemented with glycerol so that the layers in the cassette will not mix.

In principle a concentration gradient is poured. For casting linear gra-

dients the law of the communicating vessels is followed: when the

level in one of the tubes decreases, liquid will flow through the con-

The mode of functioning of the

stacking gel is in detail

described in “Electrophoresis in

Practice” fourth edition 2004,

page 34ff.

For 2-D electrophoresis in

vertical systems the stacking gel

is not necessary.

The easiest is to use gravitation

to create the liquid flow; the

velocity can be controlled by

placing the gradient maker on

a certain level and opening the

clamp only partly. In some

laboratories the support of a

peristaltic pump is preferred.

1.2 Polyacrylamide Gel Electrophoresis 25

necting channel until the level is equal. The light solution will be

immediately mixed with the heavier solution in the mixing chamber.

Figure 1.3 shows casting of a single gel in two different ways: on the

left hand side the solution is poured from the top of the cassette. In

this case the mixing chamber contains the dense solution, the reser-

voir the light solution. A compensation rod is placed into the reser-

voir for volume and density compensation. Alternatively the mono-

mer solution can be introduced through the bottom, then the solu-

tions are swapped, the compensation bar is not inserted (see right

hand side of Figure 1.3). This option is also preferred for casting mul-

tiple gels.

Exponential gradient gels are prepared by placing a stopper into

the mixing chamber, thus keeping the volume of the mixing chamber

fixed. Exponential gradients are not often used.

In the field of proteomics gradient gels are used as immobilized

pH gradients for isoelectric focusing, or as porosity gradients in Blue

Native Electrophoresis, and sometimes for SDS gel electrophoresis.

Fig. 1.3: Preparing a linear acrylamide gradient.

Left side: pouring from the top. In the beginning the mixing

chamber contains only dense solution, when the liquid level

decreases; light solution from the reservoir flows in and is mixed

with the liquid in the mixing chamber.

Right side: pouring from the bottom. The solutions are swapped;

the gel solution is introduced through a hole in the bottom of

the casting stand. The magnetic bar is rotated with a magnetic

stirrer (not shown).

This procedure is described in

“Electrophoresis in Practice”

fourth edition 2004, page 37.

1 Electrophoretic Techniques26

1.2.1.5 Gel Sizes

Proteomics requires large gels, because highly complex protein mix-

tures have to be separated as efficiently as possible. Large gels also

offer more space for highly abundant proteins; they have a higher

dynamic range. Some expert laboratories work with gel sizes of

4030 cm (Klose and Kobalz, 1995). But in general, the standard size

for a large 2-D gel is 2520 cm, because there are upper limits for

practically handling the gels. Also, the prices for scanners increase

exponentially, when a size of letter format or A4 is exceeded. For verti-

cal systems mostly the gels are 1 mm thick. Some laboratories prefer

1.5 mm thick gels because those have a higher mechanical stability.

However, staining of these thicker gels takes longer and is in some

cases less sensitive. Horizontal gels are usually 0.5 mm thin.

There are two other standard gel sizes for vertical systems:

1616 cm and 88 cm (minigels); these gels are much easier to han-

dle, however for 2-D electrophoresis they suffer from limited resolu-

tion. Their major application area in proteomics is 1-D SDS electro-

phoresis and blotting. For 1-D separations in horizontal flatbed sys-

tems 25 cm wide and 11 cm long gels are used for running multiple

samples.

1.2.1.6 Mechanically Supported Polyacrylamide Gels

Although polyacrylamide gels have a much better mechanical stabi-

lity than agarose gels, they can swell, shrink, and become fragile after

they have been removed from the cassettes. Particularly during stain-

ing gels can easily break into many pieces. Some laboratories use

therefore gel strengtheners, which are either acrylamide derivatives

or supplements to acrylamide monomer solutions.

As an alternative, gels can be bound to one of the cassettes glass

plates, which has been pretreated with Bind-Silane. This procedure is

particularly useful, when selected bands or spots have to be cut out

with an automated spot picker. In this case the spot picker cuts the

spots from the gel while using the information on the spot position

coordinates from the image analysis results. This concept would not

work, when the gel would shrink or swell between scanning and spot

picking.

Instead of binding a gel to a glass plate, it can be cast on a film sup-

port. This has become a standard for gels, which are run on a hori-

zontal flatbed apparatus, but is also applied for vertical gels. Lately

new supporting films have been developed, which do not exhibit

fluorescent background anymore. Furthermore these films are trea-

ted with a novel binding chemistry, which does not increase the gel

pore sizes near the film surface.

Klose J, Kobalz U. Electrophor-

esis 16 (1995) 1034–1059.

Nevertheless many laboratories

are satisfied with the resolution

of 2-D separations in smaller

gels.

Gels with improved mechanical

strength and elasticity are

obtained with these reagents,

however on cost of resolution

and pattern quality.

If the gel has to be stained, the

reagents and dyes can diffuse

into the gel only from the open

side, which can lead to longer

staining time and to lower

sensitivity, for instance for silver

staining.

Gels on film supports are easier

to ship, handle and store than

glass plate supported gels.

1.2 Polyacrylamide Gel Electrophoresis 27

1.2.1.7 Reversible Polyacrylamide Gels

Practical experiences in mass spectrometry analysis of peptide mix-

tures show that better signals are obtained from samples digested in

liquid phase rather than in gel plugs. Besides methylenebisacryla-

mide a number of other cross-linking reagents exist, listed and com-

pared by Righetti (1983). Some of them possess a cleavage site, which

allows solubilization of the gel matrix after electrophoresis (see

Table 1.1).

Tab. 1.1: Alternative crosslinkers for polyacrylamide gels (examples).

Substance Cleavage site Cleavage agent Comment

N,N¢-methylenebisacrylamide

(Bis)

None Not possible Standard crosslinker

N,N¢-(1,2-Dihydroxyethylene)bisacrylamide

(DHEBA)

1,2-diol Periodic acid

N,N¢-Diallyl-ditartardiamine

(DATD)

Ester bonds Hydrolysis with

a base

Problems with inefficient

polymerization

N,N¢-Bisacryloylcystamine

(BAC)

Disulfide bond Thiol reagents DTT in the sample must

be completely scavenged

by iodoacetamide

Furthermore, reversible polyacrylamide gels can become very inter-

esting as a sample preparation method for the “top-down” approach

in proteomics, where intact proteins are directly analyzed with very

high resolution mass spectrometry like FT-ICR and orbitraps (see

page 232 f).

1.2.2

SDS Polyacrylamide Gel Electrophoresis

In general this is the mostly applied electrophoretic method for pro-

tein analysis. In proteomics SDS PAGE of proteins is employed for

many different purposes:

Second dimension in high-resolution 2-D elec-

trophoresis;

1-D protein separation prior to tryptic digestion

and LC-MS of peptides;

Second dimension for “dual detergent” electro-

phoresis (after separation with 16-BAC or

CTAB);

Second dimension for Blue native electrophor-

esis;

Third dimension in IPAS.

Righetti PG.: Isoelectric

focusing: theory, methodology

and applications. Elsevier

Biomedical Press, Amsterdam

(1983).

1 Electrophoretic Techniques28

1.2.2.1 Theoretical Background

Sodium dodecyl sulfate (SDS) is a very strong anion detergent, and

solubilizes all proteins including the very hydrophobic ones. It dena-

tures proteins by dissolving hydrogen bonds and unfolds the tertiary

and secondary structures. SDS and proteins form complexes with a

necklace-like structure composed of protein-decorated micelles con-

nected by short flexible polypeptide segments (Ibel et al. 1990). As a

result of the necklace structure large amounts of SDS are incorpo-

rated in the SDS–protein complex in a ratio of approximately 1.4 g

SDS/g protein. SDS masks the charge of the proteins themselves and

the formed anionic complexes have a reasonably constant net nega-

tive charge per unit mass. Usually a reducing agent such as DTT is

added to the sample to cleave the disulfide bridges between cysteines.

Then the polypeptides become completely unfolded, all quaternary

structures are dissolved.

With SDS PAGE (SDS–polyacrylamide gel electrophoresis) the

polypeptides are separated according to their molecular weights (M

All proteins – also those with basic pIs – will migrate towards the

anode. The electrophoretic mobility of proteins treated with SDS and

DTT depends largely on the molecular weight of the protein. At a cer-

tain polyacrylamide percentage there is an approximately linear rela-

tionship between the logarithm of the molecular weight and the rela-

tive migration distance of the SDS–polypeptide complexes of a cer-

tain molecular weight range. The molecular weights of the sample

proteins can be estimated with the help of co-migrated standards

with known molecular weights.

& Note: SDS PAGE cannot deliver the exact

molecular mass of a protein, it allows only

estimation.

1.2.2.2 Sample Preparation

In the standard sample preparation procedure for 1-D SDS electro-

phoresis the proteins are boiled for 3 minutes in the sample buffer

containing 2% SDS, 50 mmol/L Tris-Cl pH 8.8, 0.01% Bromophenol

blue, and 1% (w/v) DTT. For vertical gels the sample buffer must con-

tain 25% glycerol to prevent mixing of the sample with the upper buf-

fer. When the sample has cooled down, back folding and aggregating

of subunits must be prevented: either by adding the same amount of

reductant again or by alkylating the cysteines with 2.5% (w/v) iodo-

acetamide. Alkylation has several advantages: higher stability of the

samples, prevention of artifactual lines across the gel and for down-

Ibel K, May RP, Kirschner K,

Szadkowski H, Mascher E,

Lundahl P. Eur J Biochem 190

(1990) 311–318.

For complex protein mixtures

mostly gels with T values of 12

to 13 are used for the optimal

separation in the range

between 10 kDa and 100 kDa.

Exact masses can only be deter-

mined with mass spectrometry.

It should be noted that during

electrophoresis the polypeptides

become partly alkylating by

acrylamide. For database

searches after mass spectro-

metry it is very important that

either none or all cysteines are

alkylated.

1.2 Polyacrylamide Gel Electrophoresis 29

stream analysis with mass spectrometry ensuring alkylation of all

cysteines.

For some clinical applications, or for the detection of antibodies by

blotting, sometimes non-reduced samples are applied, in order to

maintain the quaternary structure of the immunoglobulins. In this

case the molecular weights cannot be determined, because the disul-

fide bridges are still intact, many of the polypeptides are still folded.

Non-reduced samples must not be boiled; this would cause fragmen-

tation of some polypeptides.

1.2.2.3 Buffers and Gels

Tris-chloride / Tris-glycine

The standard buffer system for SDS

PAGE is based on the discontinuous Tris-chloride / Tris-glycine sys-

tem described by Laemmli (1970). With this buffer system, reproduci-

ble and well resolved patterns are obtained even with high protein

loads. It should be noted that different laboratories prepare the buf-

fers in slightly different ways, this can cause differences of the run-

ning conditions. When the gel buffer is titrated with a pH meter, the

measuring electrode should be well calibrated. Some laboratories

measure the added hydrochloric acid by the volume.

The running buffer and the gels contain 0.1% SDS. Sometimes the

gels are cast without SDS, because the SDS migrating into the gel

from the cathodal buffer is sufficient. During electrophoresis the

negatively charged chloride, SDS and glycine ions migrate towards

the anode, the positively charged Tris ions migrate towards the cath-

ode. The buffer reservoirs of the electrophoresis chamber must be

large enough to prevent depletion of the buffer ions.

& Note: Use only Tris base for making the buffers,

not “Tris buffer”. The gel buffers are made with

Tris and HCl, they contain only chloride. The

cathodal running buffer must not contain any

chloride; only SDS, Tris and glycine. Do not

titrate the upper running buffer to adjust the

pH!

Because of the very alkaline pH 8.8 in the gel of the Laemmli system,

the polyacrylamide matrix becomes hydrolyzed during storage. After

two months the sieving property of the matrix is almost completely

gone. In the standard laboratory practice this is not a big issue,

because the gels are consumed within one week after preparation.

The situation is different, when ready-made gels are used.

Some people call this “native”

SDS electrophoresis.

Lmmli UK. Nature 227

(1970) 680–685.

Adding HCl to the upper

running buffer eliminates the

stacking effect and causes

extended running time.

The problem is not only the

limitation in shelf life, but also

the lack of reproducibility

because of the ongoing hydro-

lysis of the matrix.