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

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1 Electrophoretic Techniques30

Long shelf life SDS gels For long shelf life, the pH value of the gel

buffer has to be reduced to or below pH 7. This requires a modifica-

tion of the buffer composition.

Tris-acetate / Tris-tricine Tris-acetate buffer with a pH of 6.7 has pro-

ven to have a good storage stability and separation capacity for flatbed

gels. Glycine has to be replaced by tricine as the terminating ion.

Since tricine is more expensive, it is only used in the cathodal buffer.

The anodal buffer contains Tris-acetate. The running buffer can be

applied as polyacrylamide electrode strips, instead of connecting the

gels to liquid buffers in tanks, see also page 108. The molecular weight

distribution of proteins obtained with this buffer system is slightly

different from that achieved with the Laemmli system. This buffer

system is also applied by rehydratization of pre-polymerized, washed

film-supported gels for SDS in horizontal flatbed chambers (Clean-

Gel). Figure 1.4 shows a SDS electrophoresis result in a 10% T gel.

Fig. 1.4: SDS electrophoresis in a CleanGel 10% T.

Samples: plant seed proteins and M

standards.

Hot Coomassie brilliant blue staining.

Bis-Tris / Bis-Tris-tricine This is similar to the above system. Tris is

replaced by Bis-Tris to obtain improved buffer capacity at neutral pH

(Wiltfang et al. 1991). Also with this buffer system the spot patterns

are different from Laemmli buffer patterns.

Because of the tricine in the

cathodal buffer, these gels show

a very good separation of small

peptides, when a gradient gel or

a 15% T gel is used.

Wiltfang J, Arold N, Neuhoff V.

Electrophoresis 12 (1991)

352–366.

1.2 Polyacrylamide Gel Electrophoresis 31

PPA-chloride / Tris-glycine Here the Tris in the gel is replaced by

PPA (piperidino propionamide) and titrated with hydrochloric acid to

pH 7. This gel buffer can be combined with the standard running

buffer Tris-glycine in the cathodal buffer. For the anodal buffer PPA

would be required instead of Tris. Because this compound is rather

expensive, the large volume anodal buffer usually contains a cheaper

compound: diethanolamine-acetate (DEA).

Tris-borate for improved separation of glycoproteins Glycoproteins

migrate slower than pure polypeptides, since the sugar moiety does

not bind SDS. When a Tris-borate-EDTA buffer is used, borate sticks

to the sugars, the sugar moieties become also negatively charged:

therefore the speed of migration will increase (Poduslo, 1981). The

glycine in the running buffer is simply replaced by boric acid. The

use of gradient gels is also beneficial for better MW estimations. See

also cationic detergent electrophoresis on page 34 f.

Homogeneous gels Usually a homogeneous gel with 12.5% T and

3% C is used; the proteins of major interest in the size range from 10

kDa to 100 kDa are optimally resolved. Only in special cases the

matrix concentration is modified to increase the resolution in certain

molecular size ranges.

The rule of thumb is:

Lower T value: better resolution for high molecu-

lar weight proteins.

Higher T value: better resolution for low molecu-

lar weight proteins.

Gradient gels Gradient gels offer a broader separation interval than

homogeneous gels, also the linear relation interval between the loga-

rithm of M

and the migration distance is wider than for homoge-

neous gels. And they exhibit a zone sharpening effect.

Peptide gels The resolution of peptides below 10 kDa is not suffi-

cient in conventional Tris-chloride / Tris-glycine systems. The pep-

tides smaller than 10 kDa co-migrate with the SDS front. A number

of modifications of the standard gel and buffer system have been pro-

posed, for instance the addition of 8 mol/L urea to the gel by Hashi-

moto et al. (1983), and, additionally, increasing the Tris concentration

in the gel to 1 mol/L by Anderson et al. (1983). The most efficient

technique has been developed by Schgger and von Jagow (1987):

The resolving gel has a composition of 16% T and 6% C; the gel buf-

fer concentration is increased to 1 mol/L Tris-chloride; the pH is low-

ered to 8.4; and tricine is used as terminating ion instead of glycine.

In this way the destacking of peptides and SDS is much more effi-

The M

distribution is very

similar to Laemmli gels.

Poduslo JF. Anal Biochem 114

(1981) 131–139.

Hashimoto F, Horigome T,

Kanbayashi M, Yoshida K,

Sugano H. Anal Biochem 129

(1983) 192–199.

Anderson BL, Berry RW, Telser

A. Anal Biochem 132 (1983)

365–275.

Schgger H, von Jagow G. Anal

Biochem 166 (1987) 368–379.

1 Electrophoretic Techniques32

cient than in the standard Tris-glycine system. This method yields lin-

ear resolution from 100 kDa to 1 kDa.

The Schgger system requires long running time because of the

high buffer concentration in the gel, which would otherwise become

overheated with high electric power. By adding 30% v/v ethylenegly-

col to the monomer solution, the buffer concentration can be reduced

to 0.7 mol/L (Westermeier, 2004). The resolution is still very good

and cracking of glass plates and smiling effects are avoided.

1.2.3

Blue Native Electrophoresis

Blue Native polyacrylamide electrophoresis has been developed by

Schgger and von Jagow (1991) for the separation of membrane pro-

teins and hydrophobic protein complexes in enzymatically active

form. Membrane proteins and complexes are extracted with the help

of a mild non-ionic detergent like Triton X-100, dodecyl-b-D-malto-

side, or digitonin. The gel buffer is composed of e-amino caproic acid

and Bis-Tris and titrated to the physiological pH 7.4. The anode buffer

is Bis-Tris titrated to pH 7.0 with HCl, the cathodal buffer tricine, Bis-

Tris and Coomassie blue dye. The anionic dye Coomassie brilliant

blue G-250 is added to the sample and the cathodal buffer. The dye

binds to the hydrophobic proteins and complexes without disturbing

protein–protein interactions and provides negative charges to them

independently of their original net charge. Thus under the mild con-

dition of pH 7.4 all sample components migrate into the anodal direc-

tion, and they are visible during the separation. In fact the dye–pro-

tein complexes are soluble in the detergent-free buffer medium.

Because all protein and complex surfaces are negatively charged, they

repel each other, and they will not aggregate. In the middle of the

run, the cathodal buffer is exchanged with a non-dye-containing buf-

fer, in order to achieve a clear background. The gels do not need to be

stained, because the Coomassie dye is still bound to hydrophobic pro-

teins and the complexes. Valuable hints for performing Blue native

PAGE including a trouble shooting guide can be found in a recently

published review by Wittig et al. (2006).

Porosity gradient gels from 4.5% T to 16% T are used, to allow large

super-complexes to enter and to prevent smaller molecules and com-

plexes to leave the gel on the end. Very sharp zones can be observed,

because the complexes migrate until the gel network gets too tight

for further migration. The working range is between 10 kDa and

10,000 kDa. A stacking gel with the same buffer, but with a slightly

lower concentration than the end of the gradient of 4% T is used for

optimum sample entry.

Westermeier R. Electrophoresis

in Practice. WILEY-VCH, Wein-

heim (2004) 242–245.

Schgger H, von Jagow G. Anal

Biochem 199 (1991) 223–231.

Wittig I, Braun H-P, Schgger

H. Nat Protocols 1 (2006)

419–428.

Blue Native electrophoresis is

often run in minigels or

medium gel format. In the

proteomics environment large

gel systems are preferred with

2520 cm gels in the second

dimension.

1.2 Polyacrylamide Gel Electrophoresis 33

In order to determine the complex partners the lanes are cut out,

these polyacrylamide strips are equilibrated in SDS buffer and placed

on top of a SDS gel. Thereby the complexes become dissociated into

the single proteins, which are separated in a second dimension.

Because in SDS electrophoresis the Coomassie dye will separate from

the proteins and migrate in the front, the gels need to be stained

afterwards, which is mostly performed with Coomassie blue (Gran-

vogl et al. 2006).

Figure 1.5 shows an example of two-dimensional electrophoresis

employing Blue Native electrophoresis in the first dimension (from

Eubel et al. 2003) and SDS electrophoresis in the second.

Fig. 1.5: Blue Native PAGE / SDS PAGE electrophoresis of mitochondrial

complexes of Arabidopsis thaliana (from Eubel et al. 2003). The complexes

were solubilized with digitonin and they are annotated with Roman numerals.

I+III is a super complex composed of complexes I and III. Coomassie brilliant

blue post-staining of the SDS gel.

The technique is applied in two major areas of proteomics:

Analysis of very hydrophobic proteins, like mem-

brane proteins.

Functional proteomics: analysis of protein com-

plexes and super-complexes.

A comprehensive review on applications of Blue native PAGE can be

found in the paper by Krause (2006).

For differential analysis of complexes it is very useful to apply the

DIGE concept. The complexes can easily be labeled with the CyDyes

after solubilization, prior to adding the Coomassie dye (Perales et al.

2005).

Granvogl B, Reisinger V,

Eichacker LA. Proteomics 6

(2006) 3681–3695.

Eubel H, Jnsch L Braun H.

Plant Physiol 133 (2003)

274–286.

Krause F. Electrophoresis 27

(2006) 2759–2781.

Perales M, Eubel H, Heine-

meyer J, Colaneri A, Zabaleta

E, Braun H-P. J Mol Biol 350

(2005) 263–277.

1 Electrophoretic Techniques34

When digitonin is used for extraction, even the big super-com-

plexes are kept together and intact during the separation. After cut-

ting out the lanes and equilibration in a less mild detergent, namely

dodecyl maltoside containing buffer, the single complexes can be

separated in a second-dimension Blue Native gel. In the resulting pat-

tern the composition on the super-complexes is displayed. With activ-

ity staining, a zymogram technique, its function can be assigned to

the particular spot. All this is nicely demonstrated in a paper by Sun-

derhaus et al. (2006). In this paper these complexes are also directly

visualized by single particle electron microscopy.

Three-dimensional electrophoresis The analysis of smaller subsets of

protein mixtures frequently provides improved pattern and allows to

detect proteins which otherwise do not enter the gel. A possible pro-

cedure is the three-dimensional electrophoresis procedure introduced

by Werhan and Braun (2002). The first dimension is the separation of

protein complexes by blue native polyacrylamide electrophoresis. The

visible bands are eluted electrophoretically from the gel, destained,

and further analyzed by standard denaturing 2-D electrophoresis:

IEF/SDS PAGE. Interestingly, this method reveals also proteins with

high hydrophobicity; most probably due to the considerable reduction

of complexity of the protein mixture.

Stegemann et al. (2005) have introduced a three-dimensional gel

block for performing blue native electrophoresis and SDS electro-

phoresis in one go.

1.2.4

Cationic Detergent Electrophoresis

Another possibility for getting poorly soluble proteins separated and

analyzed is the combination of two electrophoretic separations in pre-

sence of two different ionic detergents. Usually the extraction and

separation with a cationic detergent is selected as the first dimension,

because cationic detergents are less denaturing than SDS. The pro-

tein–detergent micelles migrate towards the cathode. The separation

pattern is different from that obtained with SDS electrophoresis. Pro-

teins are either solubilized with cetyltrimethylammonium bromide

(CTAB) or benzyldimethyl-n-hexadecylammonium chloride (16-BAC)

and applied on an acidic polyacrylamide gel containing the respective

detergent. A sodium or potassium phosphate buffer pH 2.1 with a

stacking gel pH 4.1 is used. It has been observed that membrane gly-

coproteins are separated much better in acidic CTAB electrophoresis

than in basic SDS electrophoresis (Buxbaum, 2003).

Sunderhaus S, Dudkina N,

Jnsch L, Klodmann J, Heine-

meyer J, Perales M, Zabaleta

E, Boekema E, Braun H-P.

J Biol Chem 281 (2006)

6482–6488.

This procedure can also be cate-

gorized into pre-fractionation

with Blue native electrophoresis

followed by 2-D electrophoresis.

Werhahn W, Braun H-P. Elec-

trophoresis 23 (2002) 640–

646.

Stegemann J, Ventzki R,

Schrodel A, de Marco A.

Proteomics 5 (2005)

2002–2009.

Buxbaum E. Cationic electro-

phoresis and electro transfer of

membrane glycoproteins. Anal

Biochem 314 (2003) 70–76.

1.3 Blotting 35

For 2-D electrophoresis the first dimension is either run in indivi-

dual gel rods in thin glass tubes or carried out in slab gels, which are

cut into strips for the second dimension (see Figure 1.6). After the

first dimension the strip is equilibrated in SDS sample buffer and

applied on the second-dimension gel (MacFarlane, 1989).

Fig. 1.6: Schematic representation of 2-D electrophoresis with

cation detergent / SDS PAGE. Here the first dimension is performed

in a vertical slab gel.

Hartinger et al. (1996) were the first to use the technique to success-

fully separate integral membrane proteins. Usually the patterns

achieved with this technique are not easy to compare. However, when

the samples are prelabeled with different CyDyes, mixed and applied

together on the gel, the same proteins of the different samples will

co-migrate, and the patterns can be analyzed with a spot co-detection

algorithm. It was shown by Helling et al. (2006) that very scarce sam-

ples can be analyzed in this way by pre-labeling the cysteines of the

proteins with saturation dyes. In this approach the first dimension

was performed with CTAB in narrow gel rods instead of slab gels.

1.3

Blotting

Blotting is the transfer of macromolecules on immobilizing mem-

branes for specific and sensitive detection. The electrophoretic trans-

fer of electrophoretically separated proteins onto a membrane with

subsequent immuno detection was introduced by Towbin et al.

(1979). This method is frequently called “Western blotting”. For pro-

tein blotting either nitrocellulose or PVDF is used as membrane

material. PVDF membranes have a higher binding capacity for pro-

teins than nitrocellulose, but nitrocellulose binds small proteins

better.

Large gel formats are preferred

for this technique, because the

spots are crowded along the

diagonal axis and require as

high resolution as possible.

MacFarlane DE. Anal Biochem

132 (1983) 231–235.

Hartinger J, Stenius K, Hoge-

mann D, Jahn R. Anal

Biochem 240 (1996) 126–133.

Helling S, Schmitt E, Joppich

C, Schulenborg T, Mllner S,

Felske-Mller S, Wiebringhaus

T, Becker G, Linsenmann G,

Sitek B, Lutter P, Meyer HE,

Marcus K. Proteomics 6 (2006)

4506–4513.

Towbin H, Staehlin T, Gordon

J. Proc Natl Acad Sci USA. 76

(1979) 4350–4354.

The term Western Blotting is

derived from the name of

Edwin Southern, who intro-

duced the first blotting tech-

nique: the transfer of DNA

onto nitrocellulose by capillary

forces for subsequent hybridiza-

tion (Southern EM. J Mol Biol.

98 (1975) 503–517).

1 Electrophoretic Techniques36

Usually SDS polyacrylamide electrophoresis is employed for the

separation, because all proteins have been solubilized and migrate in

the same direction, and the epitopes are easier accessible due to the

denaturing effect of SDS. Proteins are too big to elute quantitatively

from a SDS polyacrylamide gel by diffusion or capillary forces, there-

fore they are transferred electrophoretically.

1.3.1

Electrophoretic Transfer

As shown in Figure 1.7 there are several ways to perform the electro-

phoretic transfer: tank blotting, semi-dry blotting and semi-wet blot-

ting. All three have in common that the gel and the membrane form

a sandwich with a stack of filter papers on both sides.

Fig. 1.7: Blotting transfer methods.

If isoelectric focusing is used for the separation of the proteins, it is

more efficient to transfer the proteins by diffusion with pressure blot-

ting (Towbin et al. 2001).

1.3.2

Protein Detection on the Membrane

After the transfer, which can take about an hour (semidry blotting) to

overnight (tank blotting), the free binding sites of the membrane is

blocked with a protein mixture, which will not interfere with the sub-

sequent probing with an antibody. After some washes this “primary

antibody” is detected with a secondary antibody, which recognizes

this particular antibody. This secondary antibody is conjugated with a

set of specific molecules, which can be easily detected with a subse-

quent development procedure with high sensitivity. The most sensi-

tive detection methods are using enhanced chemiluminescence

(ECL): the antibody–horseradish conjugate recognizes the primary

antibody; the substrate reaction is coupled with a secondary reaction

For blotting small or medium

sized gels are preferred, in this

way the volume of expensive

antibody solutions is kept to a

minimum.

Towbin H, zbey , Zingel O.

Electrophoresis. 22 (2001)

1887–1893.

More detection techniques are

described in “Electrophoresis in

Practice”.

1.3 Blotting 37

which causes chemiluminescent light emission for a certain time per-

iod. This light signal is accumulated by exposing the membrane on

an X-ray film, or by placing it into an absolutely dark cabinet where

the signal is recorded with a sensitive CCD camera. With a special

variant of ECL down to 1 pg of a protein band is detectable. Figure 1.8

shows an example for detection of a single protein with ECL versus a

Coomassie blue-stained gel showing all proteins.

Fig. 1.8: Comparison of a gel with total proteins Coomassie

blue-stained versus a blot membrane with one protein detected

with ECL. Note the high sensitivity of blotting detection.

The proteins on a blotting membrane can be reprobed for several

times after stripping the antibodies off and washing the membrane.

For this case it is recommended to use PVDF membranes or sup-

ported nitrocellulose.

Multiplex detection on blots As an alternative to enzyme–substrate

detection methods fluorescent dyes, like CyDye, can be conjugated to

the secondary antibody. In this case the fluorescent signal is directly

recorded with a fluorescent scanner. This procedure offers almost the

same sensitivity like enhanced chemiluminescence, and with differ-

ent fluorescence-conjugated secondary antibodies several different

antigens can be probed in one procedure without the need for strip-

ping antibodies off from the membrane. Besides multiplex detection

this allows also a relative quantification of changes of expression

levels of an antigen, when the second primary antibody is directed

against a protein with stable expression level.

Blotting from 2-D electrophoresis gels There are many papers pub-

lished where immunoblotting was successfully performed after 2-D

For this method low fluorescent

membranes must be used.

Blotting from 2-D electrophor-

esis gels had been the standard

for protein identification, until

mass spectrometry became so

sensitive that it detected too

many constituents of the blot-

ting membranes.

1 Electrophoretic Techniques38

electrophoresis. However, it is frequently observed that an antibody

does not recognize a protein anymore, when the sample had been

separated with 2-D electrophoresis; but the same protein can easily

be detected on a blot after 1-D SDS electrophoresis. It is still not

known, what causes this effect. There is a hypothesis that focusing a

protein at its isoelectric point and/or the presence of high molar urea

in the first dimension causes a different and stronger denaturation

for some proteins than SDS treatment alone.

Non-immunological detection procedures There are a number of

general protein staining techniques for blot membranes, which are

reversible or will not interfere with the subsequent specific detection

methods. The most practical are the fluorescent dyes, which are intro-

duced below in the context of staining of 2-D gels on page 119 ff.

Specific proteins can be detected with other ligands than antibo-

dies. For instance, glycoproteins are detected with high specificity

using lectins coupled to very sensitive avidin–biotin detection proce-

dures. This method is the most reliable specific detection of glycosy-

lated proteins, as the glyco-specific staining methods often produce

some wrong positive signals. For the detection of protein–protein

ligands there is a method called “Far Western Blotting”: The probing

protein is tagged with a labeled antibody for detection (Burgess et al.

2000).

In proteomics blotting is mostly employed for the verification of a

biomarker or drug target. When the new approach of antibody proteo-

mics has succeeded with delivering antibodies against each human

protein, blotting will gain increasing importance for the identification

of very low abundant proteins.

1.4

Isoelectric Focusing

In proteomics isoelectric focusing is mainly applied for the following

purposes:

First dimension in high-resolution 2-D electro-

phoresis of complex protein mixtures.

Pre-fractionation of complex protein mixtures

according to charge.

For the separation of very heterogeneous mix-

tures of tryptic peptides instead of strong cation

exchange chromatography in MDLC-MS.

Burgess R, Arthur TM, Pietz

BC. Methods Enzymol. 328

(2000) 141–157.

The application of blotting for

N-terminal sequencing or mass

spectrometry analysis has

almost been completely aban-

doned.

1.4 Isoelectric Focusing 39

1.4.1

Theoretical Background

Isoelectric focusing is performed in a pH gradient. Proteins are

amphoteric molecules with acidic and basic buffering groups. Those

become protonated or deprotonated depending on the pH environ-

ment. In basic environment the acidic groups become negatively

charged, in acidic environment the basic groups become positively

charged. The net charge of a protein is the sum of all negative or posi-

tive charges of the amino acid side chains. When net charges of pro-

teins are plotted over the pH scale, each protein has an individual net

charge curve. The intersection of the net charge curve with the x axis

is the isoelectric point – the pH value where the net charge is zero.

This is shown for two model proteins in Figure 1.9.

When a protein is applied on a certain pH value of the gradient,

and an electric field is applied, it will start to migrate towards the elec-

trode of the opposite sign of its charge. Because it migrates inside a

gradient, it will arrive at a pH value of its isoelectric point (see Figure

1.9) after some time. At its pI it has no net charge anymore and stops

migrating. Should it diffuse away above or below its pI it will become

charged again and migrate back to its pI. This is called the “focusing

effect”, which results in very high resolution.

Fig. 1.9: The principle of isoelectric focusing.

Left: Net charge curves of two model proteins A and B. At the point of

application, A will have two positive, B will have one negative charge(s).

Right: Migration of A and B to their pIs in the pH gradient of an isoelectric

focusing gel.

& In principle: Isoelectric focusing is a very high

resolution separation method, and the pI of a

protein can be measured.