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

Подождите немного. Документ загружается.

1 Electrophoretic Techniques110

sample: The spot patterns are very similar. It is demonstrated that

sharper spots are obtained in thinner gels, which can be employed in

the flatbed system, but not in a vertical setup.

Design of vertical instruments The following functions are needed in

the instrument for the second dimension:

High throughput;

Large gel cassettes;

Efficient cooling for fast runs with straight front;

Leakage free;

Reproducible results independent from gel posi-

tion;

Different gel thickness should be possible;

Easy handling;

Buffer consumption not too high.

The first high-throughput chamber had been developed by Anderson

and Anderson (1978): the DALT box, where the gel cassettes are tilted

by 90 degrees and inserted between insulating rubber flaps (see Fig-

ure 1.42). In contrast to conventional vertical systems, the migration

direction of the proteins is from left to right instead of from the top

to the bottom. Handling of the instrument is easy. As only one single

tank is used, there is no problem of buffer leakage from an upper to a

lower tank.

Fig. 1.42: Schematic drawing of the traditional DALT chamber

for multiple SDS PAGE runs. The migration direction is from the

left to the right. Insulating rubber flaps prevent current flow

between the cassettes.

The gels are usually run overnight, because the cooling efficiency

is limited. The entire box is filled with Tris-glycine buffer. The buffer

Anderson NG, Anderson NL.

Anal Biochem 85 (1978)

341–354.

The buffer volume of 20 L is

rather high.

1.5 Two-dimensional Electrophoresis 111

should not be used more than once, because it will become enriched

with chloride ions from the gel buffer. When the Tris-glycine buffer

contains chloride ions, the initial stacking effect is disturbed – result-

ing in a loss of resolution – and the running time is extended.

A limitation is the design of the buffer tank: the cathodal and ano-

dal buffer reservoir is identical. This makes peptide separations with

Tris-tricine buffer very expensive. And – the stable PPA buffer system

cannot be applied, because it would require two completely separate

tanks for the anodal and the cathodal buffer.

For vertical instruments with completely separate tanks, several

construction concepts are possible. Figure 1.43 shows three different

designs.

Fig. 1.43: Three different designs of vertical chambers.

All of them can be cooled.

A. Buffer-backed chamber.

B. Chamber with tight upper buffer tank.

C. Chamber for fast multiple runs.

Vertical electrophoresis chambers

In the buffer-back chambers (Figure 1.43, type

A) the gels are cooled via the cathodal buffer.

Notched glass or aluminum oxide ceramics

plates are used to enable the contact between the

cathodal buffer and the gel.

When the buffer tanks are

completely separate, the

anodal buffer can be used

repeatedly, because chloride

ions do not disturb there.

Maximal two gels can be run;

therefore the principle is mostly

applied on miniformat appa-

ratus.

1 Electrophoretic Techniques112

The medium sized chamber with tight upper

buffer tank (Figure 1.43, type B) can accommo-

date up to four gels at the same time, when

notched glass plates are inserted between each

two gel pairs. The gels are cooled via the anodal

buffer.

Multiple runs with large gels can be performed

in chambers of type C in Figure 1.43. It is, how-

ever, important to fill upper and lower buffer to

the same level to avoid buffer mixing. The heat

removal in this chamber type is very efficient,

because the cooling anodal buffer is vigorously

pumped around in the lower tank.

The concept of pumping the anodal buffer around the cassettes is

employed for large gel systems: stand-alone instruments (Figure

1.44A) and in integrated systems for running up to 12 gels (Figure

1.44B).

Fig. 1.44: Electrophoresis instruments for large cassettes.

A: Stand-alone instrument for up to six gels.

B: Integrated apparatus Ettan DALT twelve for multiple 2-D PAGE.

The programmable power supply controls also the Peltier cooling

system and the pump for circulating the lower buffer.

At the rear of the separation unit of the integrated system sits a

draining valve for convenient removal of used buffer. At the back

panel of the control unit of the Ettan DALT twelve there is a serial port

for possible software updates and to connect a computer or serial

printer to the instrument for a report on the electric conditions after

every five minutes of the run. With this interface the instrument can

In this concept the edges of the

glass plates must be absolutely

intact to prevent leakage of the

upper tank.

The buffer concentration for the

upper tank must usually be

doubled to prevent depletion of

the cathode buffer.

1.5 Two-dimensional Electrophoresis 113

be integrated into the laboratory workflow system and allow proce-

dures according to GLP (good laboratory practice).

& Note: for optimally reproducible 2-D patterns all

gels of an experiment should be run together in

one chamber.

The following hint goes for all vertical chambers with separate tanks:

When the lower – anodal – buffer is mixed with the used upper –

cathodal – buffer after each run, the Tris concentration is high

enough for repeated use as anodal buffer. This saves work and

reagent costs. Of course, the cathodal buffer must be new for each

electrophoresis.

1.5.4.4 Equilibration of IPG Strips

Prior to the run in the second dimension the strips have to be equili-

brated with SDS buffer to transform the focused proteins into SDS–

protein complexes, which are completely unfolded and carry negative

charges only. The following conditions have been optimized by Gçrg

et al. (1987b).

& Equilibration stock solution: 2% (w/v) SDS, 50

mmol/L Tris HCl pH 8.8, 0.01% (w/v)

Bromophenol blue, 6 mol/L urea, 30% (v/v)

glycerol

Equilibration is performed twice on a shaker:

15 min 10 mL equilibration stock solution Plus 1% (w/v) DTT

15 min 10 mL equilibration stock solution Plus 2.5% (w/v) iodoacetamide

Sodium dodecyl sulfate With the amount of 2% SDS there is usually

sufficient SDS for preparative protein loads. Some laboratories use 3–

6% SDS to facilitate the migration of focused hydrophobic proteins.

Tris HCl buffer pH 8.8 In former protocols it was proposed to add the

stacking gel buffer pH 6.8, because the use of a stacking gel was stan-

dard procedure. However, it is better to add the more basic resolving

gel buffer with a pH of 8.8 to improve the alkylation of the cysteines.

Bromophenol blue This tracking dye allows a control of the running

conditions, the shape of the migration front, and the separation time.

Running gels of one experiment

in different chambers makes

spot matching more difficult.

The Tris moves from the anodal

to the cathodal buffer. Glycine

moves from the cathodal to

anodal buffer, it is not needed

in the anodal buffer.

Gçrg A, Postel W, Weser J,

Gnther S, Strahler JR,

Hanash SM, Somerlot L. Elec-

trophoresis 8 (1987b) 122–124

Note: with iodoacetamide the

solution can easily become

acidic. This would disturb the

formation of the SDS–protein

complexes.

1 Electrophoretic Techniques114

Urea and glycerol These additives have been introduced to keep

electroendosmotic effects (see above) as low as possible. Urea is also

supporting the solubility of hydrophobic proteins.

Dithiothreitol After the proteins have been focused they have to be

treated with the reductant again. When very high protein loads are

analyzed, the concentration of DTT needs to be increased.

Iodoacetamide The alkylation agent has several functions:

Complete alkylation of the cysteines, to avoid

partial modification by acrylamide for increased

spot sharpness and improved protein identifica-

tion with mass spectrometry.

Elimination of point streaking as described by

Gçrg et al. (1987b).

Avoidance of the artifactual horizontal lines

across the SDS gel in the size range of 40–

50 kDa.

When the cysteines had been pre-labeled with saturation dye for

DIGE, the iodoacetamide step is omitted.

Equilibration time Two times 15 minutes seems to be a rather long

time, and some might fear considerable losses of proteins due to dif-

fusion. As already mentioned on page 51, immobilized pH gradients

keep proteins back like a weak ion exchanger. Thus, only a little

amount of proteins – those from the surface – are washed out.

The long equilibration time is necessary for the complete formation

of SDS–protein complexes, because the negatively charged SDS is

repelled by the negative charges on the carboxylic groups of the

strips. It has been observed that a too short equilibration leads to ver-

tical streaks and losses of high molecular weight proteins.

Modified equilibration Although Hedberg et al. (2005) claim that

after running the first dimension with DeStreak under oxidative con-

dition equilibration can be shortened and both, equilibration and the

second-dimension run can be performed under oxidative conditions,

it is not recommended. This procedure is limited just to a few pH

gradients. Practice has shown that this method does not give well

resolved spot patterns for all sample types.

There are cases where increased SDS content facilitates the transfer

of hydrophobic proteins from the first to the second dimension. One

example is described by McDonough and Marbn, where 10% (w/v)

SDS in the equilibration buffer was the only way to move a highly

hydrophobic protein into the second-dimension gel.

This is valid also after running

IEF with DeStreak.

The iodoacetamide functions as

scavenger of the excess reduc-

tant.

However, the equilibration

time must not be extended too

far, because this leads to losses

of low molecular weight

proteins due to diffusion.

Hedberg JJ, Bjerneld EJ, Cetin-

kaya S, Goscinski J, Grigorescu

I, Haid D, LaurinY, Bjellqvist B.

Proteomics 5 (2005) 3088–

3096.

McDonough J, Marbn E.

Proteomics 5 (2005) 2892–

2895.

1.5 Two-dimensional Electrophoresis 115

1.5.4.5 Transfer of the IPG Strip and Running the SDS Gel

In order to make examination and evaluation of 2-D maps easier, the

first and second dimensions should always be set together in a stan-

dardized way. The majority of the scientific world agrees that it makes

sense to orientate the gel in the style of a Cartesian coordinate sys-

tem: the low values are located at the left bottom and the high values

at the right top.

& The IEF gel should always be placed on the SDS

gel in a standard orientation: low pH to the left,

high pH to the right.

Flatbed system

It is highly recommended to use the strip positioner

plate as shown in Figure 1.40A for:

Improved alignment of the electrode strips;

Preventing the electrode strips to slide away;

Reproducible positioning of the electrode strips

and the IPG strip on the SDS gel;

Achieving a straight front, because the gel sur-

face is covered.

The gels are placed directly on a cooling plate, which is connected to

a thermostatic circulator. Usually the running temperature is 15 C.

After equilibration of the IPG strip, the excess buffer is removed by

blotting it slightly with clean filter paper. The strip is placed – gel sur-

face down – parallel to and 5 mm apart from the cathodal electrode

strip. The acidic end of the strip points to the left side.

marker proteins are applied with IEF sample application pieces

as shown in Figure 1.45.

After 40 minutes of low voltage – for sample entry – the IPG strip

is removed from the gel; the cathodal electrode strip is moved from

its original position to the contact area of the IPG strip. This measure

prevents drying out and burning of the SDS gel along this contact

line. The separation can now be continued with higher voltage set-

tings.

No agarose sealing solution is

needed.

In the flatbed system EEO has

stronger effects, because the

water is transported out of the

SDS gel surface, causing drying

of the SDS gel.

1 Electrophoretic Techniques116

Fig. 1.45: Application of the IPG strip on the different types of

second-dimension gels.

Vertical system The IPG strip is placed onto the edge of the SDS gel

sideways, with its support film touching one glass plate. Hot agarose

embedding solution, pipetted onto the strip has several functions:

Achieve a gel continuity;

Prevent air bubbles between the gels;

Avoid floating of the strip in buffer solution;

Reduce electroendosmotic effects at very high

and very low pH values of the IPG strips;

Seal the gaps between cassettes spacers and the

ready-made gels.

& Note: The agarose solution should not be hotter

than 60 C; it could cause carbamylation of some

proteins because of the presence of urea.

& Composition of the agarose embedding

solution:

0.5% (w/v) agarose, SDS cathode

buffer (1 concentration), 0.01% (w/v)

Bromophenol blue

Ca. 2 mL of agarose embed-

ding solution is necessary per

gel.

1.5 Two-dimensional Electrophoresis 117

Molecular weight marker proteins.

& Tip: Best results are obtained when the

molecular weight marker proteins have the same

start conditions like the proteins from the IPG

strips: a IPG strip is rehydrated in the marker

protein solution, cut into small pieces, which can

be stored in a deep freezer or applied directly to

the SDS gel together with the IPG strips of the

first dimension.

The alternative is to apply the markers to an IEF sample application

piece directly, in a volume of 15–20 mL and let it dry.

1.5.4.6 Running Conditions for Vertical Gels

Electric conditions

Also vertical gels must be run with low voltage at

least for the first two hours to reduce the electroendosmosis effects

(see page 21). When immobilized pH gradient gels are equilibrated

in basic buffers, they become deprotonated: The carboxylic groups

become negatively charged, the amino groups are neutral. Thus the

IPG strips acquire negative net charges during equilibration with the

SDS buffer. It has been observed already during the first approaches

utilizing immobilized pH gradients in 2-D electrophoresis that this

can cause electroendosmotic effects (Westermeier et al. 1983). Those

result in protein losses due to a water flow towards the cathode. The

water transport is minimized by adding urea and glycerol to the equi-

libration solution and reducing the electric field during the phase of

protein transfer into the second-dimension gel (Gçrg et al.1985,

2003):

& Phase 1: apply only 5 mA (» 0.2 W) per gel for

2 hours

& Phase 2: continue with normal SDS

electrophoresis settings for fast or overnight runs

In discontinuous buffer systems, first the conductivity is high,

because the gel contains a lot of chloride ions, which have high mobi-

lity. When more of the glycine or tricine ions – with low mobilities –

are migrating into the gel, the conductivity decreases, and the field

strength increases. In order to avoid the production of too much Joule

heat at the end of the run, the gels are usually run at low current set-

tings. A possibility to speed up the separation is to adjust the current

setting from time to time.

Westermeier R, Postel W, Weser

J, Gçrg A. J Biochem Biophys

Methods 8 (1983) 321–330.

Gçrg A, Postel W, Gnther S,

Weser J. Electrophoresis 6

(1985) 599–604.

Gçrg A, Drews O, Weiss W. In

Simpson RJ, Ed. Purifying

proteins for proteomics: A

laboratory manual. Cold Spring

Harbor Laboratory Press, Cold

Spring Harbor, New York

(2003) 391–430.

1 Electrophoretic Techniques118

Ideally the power supply offers the feature to run the separation at

constant power. Then the current is high at the beginning and low at

the end without overheating the gels and the buffers. In this way a

separation can be run faster than under conventional conditions.

Temperature Multiple gels produce a substantial amount of Joule

heat, which has to be removed. If this is not efficient enough the gels

show the “smiling effect”: faster migration in the center than on the

lateral sides. It was the general opinion that SDS gels should be

cooled to 10–15 C to remove the heat efficiently enough to obtain a

straight front. Systematic studies in chambers with more powerful

buffer circulation have shown that better results and faster runs are

obtained when the gels are run at 25 C. Of course there are also cer-

tain limits how much temperature can be removed from the gels and

the buffers.

Benefits of quick runs Fast separations of large gels – within a few

hours instead of overnight – result in less diffused spots compared to

overnight runs. This is particularly obvious in the low molecular

weight area. Less diffused spots have three advantages:

Increased spatial resolution in homogeneous

gels;

Increased detection sensitivity;

Improved enzyme kinetics during tryptic in-gel

digestion.

1.5.4.7 Molecular Weight Determination

For the determination of molecular weights in 2-D electrophoresis

gels two different procedures are used.

Method 1. Co-running molecular weight standard proteins Molecular

weight marker proteins can be applied as a separate track to the 2-D

gel. The molecular weights of the proteins in the 2-D map are then

interpolated with the molecular weight curve obtained from the posi-

tions of the marker proteins. This procedure is usually carried out

with the image analysis software as “1-D calibration”.

The problem with this method is its limited accuracy. The markers

have to be applied at the lateral sides of the gels. Often the space is

very limited and the bands are curved because of the edge effects in

SDS PAGE. Furthermore, the estimation of molecular weights with

SDS PAGE is not accurate at all.

The high settings are applied

after the 2 hours protein

transfer.

Less need for gradient gels.

In comparative experiments

over 50% more spots could be

detected in these gels.

Because of the higher protein

concentration in the gel plug.

1.5 Two-dimensional Electrophoresis 119

Method 2. Interpolation between identified sample proteins with known

values Prominent spots showing up in each 2-D map of a sam-

ple type can be analyzed for identification and amino acid sequence

information. The theoretical M

values can then be used as keystones

for interpolating the M

values of the other proteins. The second pro-

cedure is performed with the image analysis software as “2-D calibra-

tion”. This method is much more exact than the method described

above.

1.5.5

Detection of Protein Spots

There is a demand list of properties required for the ideal spot detec-

tion technique in 2-D gels in proteomics: it should:

Be sensitive enough for low copy number

proteins;

Allow quantitative analysis;

Have a wide linearity;

Have a wide dynamic range;

Be compatible with mass spectrometry;

Be non-toxic;

Be environment-friendly;

Be affordable.

Unfortunately there is no method that affords all these features

together. Lately some reviews on detection techniques in proteomics

have been published: a “minireview” by Westermeier and Marouga

(2005), and a very elaborate compilation by Miller et al. (2006). The

following table gives an overview over currently used detection princi-

ples and their features. Several different protocols exist for most of

these methods. Those of major importance are quoted in the table.

1.5.5.1 Comparison of Detection Methods

Tab. 1.6: Detection methods for protein spots.

Method Advantages Disadvantages

Coomassie brilli-

ant blue staining

(colloidal)

Steady state method, good

quantification, inexpensive,

mass spectrometry

compatible

Medium sensitivity: LOD

ca. 20 ng of BSA; the more

sensitive, the slower

Westermeier R, Marouga R.

Bioscience Reports 25 (2005)

19–32.

Miller I, Crawford J, Gianazza

E. Proteomics 6 (2006) 5385–

5408.

Neuhoff V, Arold N, Taube D

and Ehrhardt W. Electrophor-

esis 9 (1988) 255–262.