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

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1.3 Examples 291

Tab. 1.4: Amounts of additives to 1 mL sample.

Sample 1 mL Sample 1 mL

8 mol/L urea 0.75 g 9 mol/L urea 0.94 g

0.5% (w/v) CHAPS 8 mg 2% (w/v) CHAPS 36 mg

0.28% (w/v) DTT 5 mg 1% (w/v) DTT 18 mg

1.25% (v/v) Pharmalytes 3–10*) 20 mL 2% (v/v) Pharmalytes 3–10*) 36 mL

0.002% Bromophenol blue**) 2 mL 0.002% Bromophenol blue**) 2 mL

Total volume 1.6 mL 1.85 mL

*) or IPG buffer of respective pH interval

**) from 1% Bromophenol blue solution

1.3

Examples

The volumes are given for 24 cm IPG strips only. For paper bridge

loading 18 cm strips are recommended. The sample would be diluted

in 450 mL for anodal and 350 mL for cathodal loading.

Example 1:

Escherichia coli

extract (ideal as test sample) Sample:

lyophilized cells of E. coli strain B (ATCC 11303), Sigma EC-11303

Lyophilized E. coli 30 mg Measured protein content

Lysis solution 1 mL ~12 mg protein/mL

Freeze (–20 C) and thaw two times in a microcentrifuge tube.

Centrifuge for 10 min with 13,000 rpm.

24 cm IPG strip Sample supernatant Rehydration solution

Analytical run: (~180 mg protein)

Rehydration loading 15 mL mix with 435 mL

Cup loading 15 mL*) at anodal side 450 mL (>6 h pre-rehydration)

Preparative run: (~1 mg protein)

Rehydration loading 83 mL mix with 367 mL

Cup loading 83 mL at anodal side 450 mL (>6 h pre-rehydration)

*) Mix with 50 mL rehydration solution before you apply it.

Figure 1.1 shows a typical 2-D electrophoresis pattern of an E. coli

extract applied with rehydration loading.

Step 1: Sample Preparation292

Fig. 1.1: E. coli extract (~600 mg protein), IPG DryStrip,

pH 3–10, Ettan DALT gel, fast Coomassie staining.

Example 2:

Saccharomyces cerevisiae

(yeast cell lysate)

Lyophilized yeast 300 mg

lysis solution 2.5 mL

Sonicate for 10 min at 0 C.

Centrifuge for 10 min at 10 C with 42,000 g.

24 cm IPG strip Sample supernatant rehydration solution

Analytical run:

Rehydration loading 30 mL mix with 420 mL

Cup loading 30 mL*) at anodal side 450 mL (>6 h pre-rehydration)

Preparative run:

Rehydration loading 300 mL mix with 150 mL

Cup loading 100 mL at anodal side 450 mL (>6 h pre-rehydration)

*) Mix with 50 mL rehydration solution before you apply it.

Tissue The tissue of interest is sliced with a scalpel to obtain an

appropriately sized piece. It can be frozen with liquid nitrogen and

broken into small fragments in a mortar and pestle.

1.4 Microdialysis 293

Example 3: calf liver Liver acetone powder, calf, Sigma L-7876

Acetone powder 10 mg

Lysis solution 1 mL

For small tissue amounts a disposable grinding kit is very useful

(see Figure 1.2).

Fig. 1.2: Grinding of tissue material.

Typically 5 mg of the acetone powder or 20–50 mg of animal or

plant tissue are ground in 500 mL of lysis solution.

24 cm IPG strip Sample supernatant Rehydration solution

Analytical run:

Rehydration loading 30 mL mix with 420 mL

Cup loading 30 mL *) at anodal side 450 mL (>6 h pre-rehydration)

Preparative run:

Rehydration loading 300 mL mix with 150 mL

Cup loading 100 mL at anodal side 450 mL (>6 h pre-rehydration)

*) Mix with 50 mL rehydration solution before you apply it.

1.4

Microdialysis

Dialysis of the sample is usually not recommended, because large

membrane areas can cause adsorption of proteins. Less protein gets

lost with specially designed minidialysis tubes. The fastest removal of

small ions is achieved with an 8 kDa cut-off membrane.

Salt and buffer ions can be removed efficiently by dialyzing 2-D

samples against at least 40 sample volume of 8 mol/L urea / 1%

DTT solution for 2 hours to overnight. The 2-D sample should be pre-

pared in lysis buffer. Other, more expensive additives such as CHAPS

In the Tris-glycine buffer system

peptides smaller than 10 kDa

anyhow co-migrate with the

front; they are not displayed in

the gel.

Step 1: Sample Preparation294

and carrier ampholytes do not need to be included in the dialysis

solution. These components are afterwards added back, in the origi-

nal concentrations.

1.5

Precipitation

A crude extract can contain contaminations with phospholipids and

nucleic acids, which are visualized with silver staining as horizontal

streaks in the acidic part of the gel. Protein precipitation is therefore

often employed to remove the contaminating substances. Protein pre-

cipitation is also used to concentrate proteins from samples that are

too dilute for effective 2-D analysis.

1.5.1

TCA Acetone

Sometimes the TCA and/or the salt from the sample are not comple-

tely removed, which can be observed by horizontal streaking in the

2-D gel. Practice has shown that washing with ice-cold 10% water /

90% acetone instead of pure acetone removes ionic components very

efficiently without any negative effects. Therefore the original proto-

col of Damerval et al. (1986) has been modified here. Furthermore

the modifications according to Gçrg et al. (2000) are implemented

here.

Freeze plant material – or other material – in

liquid nitrogen.

Grind it with a pestle in a pre-frozen mortar.

To 30 mg add 5 mL of 20% TCA in cold acetone

(–20 C) containing 0.2% (w/v) DTT.

Precipitate overnight in a freezer.

Centrifuge at +20 C with 13,000 rpm for

10 minutes.

wash with 90% acetone / 10% water (–20 C)

containing 0.2% (w/v) DTT.

Centrifuge again.

Resuspend the pellet in lysis buffer.

Carefully sonicate.

Centrifuge at +20 C with 13,000 rpm for

10 minutes.

For protein precipitation from a liquid the precipitation fluid must be

at least three times larger than the volume of the sample.

For precipitation of proteins

from a cell culture supernatant

often a 100% (w/v) TCA solu-

tion is employed.

Damerval C, DeVienne D, Zivy

M, Thiellement H. Electrophor-

esis 7 (1986) 53–54.

Gçrg A, Obermaier C, Boguth

G, Harder A, Scheibe B, Wild-

gruber R, Weiss W. Electrophor-

esis 21 (2000) 1037–1053.

Avoid heating!

1.5 Precipitation 295

The disadvantage is, however, that some acidic proteins get lost

with this procedure, because they are not precipitated into the pellet.

1.5.2

Ettan 2-D Cleanup Kit

The Ettan 2-D cleanup kit procedure uses a combination of a unique

precipitant and co-precipitant to quantitatively precipitate the sample

proteins. The proteins are pelleted by centrifugation and the precipi-

tate is washed to further remove non-protein contaminants. The mix-

ture is centrifuged again and the resultant pellet is resuspended into

the lysis buffer.

The sample can contain 1 mg to 1 mg protein in a volume of 1 to

100 mL. Protein can be processed from larger volumes by scaling up

the procedure.

1.5.3

Resolubilization

In practice the 2-D cleanup kit and the method according to Wessels

and Flgge hold the highest yield among the precipitation methods.

Nevertheless, there are sometimes difficulties reported to get the pel-

let back into solution. Here are a few important hints how to get the

pellet completely redissolved:

Take care that the pellet does not become com-

pletely dry!

Pipette repeatedly lysis solution over the pellet,

but do not vortex!

Resolubilization can take several hours or over-

night at room temperature. Proteases have been

completely inactivated by precipitation.

If this does not work, carefully sonicate with the

sample being placed on ice, with short bursts

and breaks in between.

Or use PlusOne Molecular grinding kit to

increase the surface of the pellet

Or freeze pellet with lysis solution at –20 C for

15 minutes.

If none of the above measures work, use SDS

solution (2% SDS, hot) and then dilute with 9

mol/L urea / 4% CHAPS to an SDS concentra-

tion below 0.1%. Also, as a rule of thumb, the

CHAPS to SDS ratio must be 8:1 in the sample

before application on the IPG strip.

This procedure has the highest

yield of precipitated proteins

and proteins coming back into

solution.

Vortexing can cause oxidation

of some proteins, and then they

will not dissolve anymore.

Do not vortex!

Avoid heating of sample

Particularly useful, when

several mg of proteins have

been precipitated.

Urea crystals will develop,

which break up the pellet

See also “SDS procedure”

below

Step 1: Sample Preparation296

1.6

Very Hydrophobic Proteins

1.6.1

Thiourea Procedure

For membrane proteins and other very hydrophobic proteins a combi-

nation urea/thiourea in the solubilization solution can be very helpful

to get more proteins into solution (Rabilloud, 1998). The modified

lysis solution is then composed as follows:

7 mol/L urea 4.2 g

2 mol/L thiourea 1.5 g

With H

dist

fill up to 9.5 mL

Removal of isocyanate:

Warm the tube to get the urea and thiourea com-

pletely in solution: Do not exceed 37 C.

Add 100 mg mixed bed ion exchanger Amberlite

IRN-150. Stir for 10 min.

Filter through paper.

Add the other additives:

4% CHAPS 400 mg

1% (w/v) DTT 100 mg

2% (v/v) Pharmalytes pH 3 to 10*) 500 mL

0.002% Bromophenol blue**) 10 mL

*) or IPG buffer

**) from 1% Bromophenol blue solution

& Important: Prepare the solution freshly, shake to

dissolve the urea, do not warm it higher than

30 C to avoid carbamylation.

This lysis solution can also be produced in larger quantities – without

DTT – and stored frozen in aliquots at a temperature lower than

–60 C.

Always treat thiourea-

containing solutions with

mixed bed ion exchanger.

Repeated freeze-thawing must

be avoided. Add DTT shortly

before use.

1.7 Quantification 297

1.6.2

SDS Procedure

Formation of oligomers can be prevented.

Organisms with tough cell walls sometimes

require boiling for 5 minutes in 1–2% SDS

before they are diluted with lysis solution.

Very hydrophobic proteins might require extrac-

tion with high percentage of SDS.

& Note: The SDS-containing sample solution must

be diluted with lysis solution (9 mol/L urea / 4%

CHAPS) to an SDS concentration below 0.1%.

Also, as a rule of thumb, the CHAPS:SDS ratio

must be 8:1 in the sample before application on

the IPG strip.

Example: human plasma According to Sanchez et al. (1995):

10 mL human plasma Mix with 6.25 mL 10% SDS, 2.3% DTT

Heat for 5 min at 95 C

Dilute with 500 mL lysis solution

& Note: SDS does not always completely separate

from the proteins during IEF, even under high

field strength. SDS can also be removed by

precipitation with the cleanup kit.

1.7

Quantification

The most reliable protein quantification for 2-D IEF samples is

obtained with the Ettan 2-D Quant kit, which is based on precipita-

tion. This procedure is compatible with such common sample pre-

paration reagents as 2% SDS, 1% DTT, 8 mol/L urea, 2 mol/L

thiourea, 4% CHAPS, 2% Pharmalytes and 2% IPG Buffer. The pre-

cipitated proteins are resuspended in a copper-containing solution;

the unbound copper is measured with a colorimetric agent. The color

density is inversely related to the protein concentration. The volume

range of the assay is 1–50 mL and the linear range for quantification

is 0–50 mg. The assay has a sensitivity threshold of 0.5 mg.

Sanchez J-C, Appel RD, Golaz

O, Pasquali C, Ravier F,

Bairoch A, Hochstrass er DF.

Electrophoresis. 16 (1995)

1131–1151.

Step 1: Sample Preparation298

1.8

SDS Samples for 1-D SDS Electrophoresis

SDS sample buffer:

2% (w/v) SDS 2 g

25% Glycerol (v/v) 85% solution 30 mL

50 mmol/L Tris HCl, pH 8.8 1.5 mol/L stock solution 3.5 mL

0.01% Bromophenol blue 10 mg

Water, deionized Make up to 100 mL

Store at room temperature.

Dissolve 50 mg of DTT in 100 mL deionized

water.

Dissolve 20 mg of iodoacetamide in 100 mL deio-

nized water.

Add 10 mL DTT solution to 100 mL SDS sample

buffer.

Mix 25 mL sample extract with 25mL SDS sample

buffer.

Heat at 60 C for 5 min.

Add 10 mL iodoacetamide solution to the 50 mL

SDS sample solution. Leave it at room tempera-

ture for 30 min.

Apply 20 mL of this solution on the SDS gel.

The glycerol is added to

increase the density to prevent

mixing with upper buffer.

Do not heat it, when the

sample contains urea!

299

Step 2: Fluorescence Difference Gel Electrophoresis

2.1

Experimental Design

The experiment setup should be properly designed: Biological repli-

cates ensure that induced biological changes are not mixed up with

inherent biological differences. Gel replicates are only necessary

when an experiment consists of a low number of samples, like two to

six samples. In this case one-time gel replicates are run, which con-

tain inverse labeled samples. Experiments with higher numbers of

samples do not require gel replicates.

Optimally all samples of an experiment should be run together in

the first and second dimension to minimize methodical variation.

This means that an experiment should not consist of more than 12

gels, if possible.

Prepare a chart for planned randomization of

the samples and the internal standard among

the gels, like the example in Table 2.1.

Tab. 2.1: Example for “randomized” sample labeling and application

for 12 samples.

Gel Cy2 standard Cy3 Cy5

1 All 12 samples Control 1 Treated 3

2 All 12 samples Control 2 Treated 1

3 All 12 samples Control 3 Treated 2

4 All 12 samples Treated 4 Control 5

5 All 12 samples Treated 5 Control 6

6 All 12 samples Treated 6 Control 4

Disposable gloves must be worn during each step to avoid contam-

ination of the sample.

Proteomics in Practice. A Guide to Successful Experimental Design 2

Ed.

Reiner Westermeier, Tom Naven, and Hans-Rudolf Hçpker

ISBN: 978-3-527-31941-1

The pooled internal standard is

always labeled with one and

the same CyDye: Cy2 for 3-

color experiments and Cy3 for

2-color experiments.

Running gels of one experiment

in different chambers makes

spot matching more difficult.

Step 2: Fluorescence Difference Gel Electrophoresis300

2.2

Sample Preparation

In the standard labeling protocol, proteins are first denatured in a

lysis buffer; that will eliminate all secondary and tertiary structures of

proteins. The protein concentration should then be determined using

the Ettan 2-D Quant kit procedure. The fluorescent properties of Cy2,

Cy3 and Cy5 can be adversely affected by exposure to light: keep the

exposure of protein labeled with CyDye to all light sources to a mini-

mum.

& During labeling the samples must not contain

primary amines (IPG buffers, carrier ampholytes)

and reductants.

Appropriate sample preparation is crucial for good results. Practice

has shown that labeling efficiency is much higher after sample

cleanup based on precipitation of the proteins. This measure will not

only remove disturbing contaminants like lipids, polysaccharides, salt

ions, and nucleic acids, but also endogeneous peptides from the sam-

ple. Peptides contain also lysines and cysteines, which would be

labeled with a CyDye fluor as well. In a standard buffer system the

small peptides migrate in the front of the second dimension, and

would not be resolved.

2.2.1

Stock Solutions

Sample lysis solutions for DIGE:

Lysis solution 1: 9 mol/L urea, 4% (w/v) CHAPS, 30 mmol/L Tris.

Lysis solution 2: 2 mol/L thiourea, 7 mol/L urea, 4% (w/v) CHAPS,

30 mmol/L Tris.

For pH adjustment: 50 mmol/L NaOH solution

Solubilize the protein samples either in sample

lysis solution 1 or 2.

Check the protein concentration in the samples:

optimally it would be between 5 mg/mL and 10

mg/mL. But 1 mg/mL to 20 mg/mL are tolerable

for successfully labeling.

Check the pH carefully: pipette 2 mL sample on a

pH indicator paper. Read out the pH value

immediately, because the color will shift with

time.

These compounds are added

after labeling had been

completed by adding a 2

sample solution.