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

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Step 6: Staining of Gels362

Fixation solution:

20% (w/v) ammonium sulfate in water.

Neutralization buffer:

0.1 mol/L Tris base, adjust pH to 6.5 with phos-

phoric acid.

6.1.1.2 Staining Procedure

Briefly wash the gel with distilled water.

Stain the gel with the staining solution overnight

or one day.

Transfer gel into neutralization buffer for 1–3

minutes.

Wash gel with 25% (v/v) methanol for less than

1 minute.

Transfer gel into fixation solution and fix pro-

teins overnight or for 1 day

For further staining, just repeat the last four

steps. Three times staining should be enough;

which takes about 1 week

& This procedure involves many steps and takes

almost a week. However, the sensitivity of the

method comes close to the sensitivity of silver

staining.

6.1.2

Sensitive “Walk-away” Coomassie Brilliant Blue Colloidal Staining

According to Anderson et al. (1991), modified by Sjouke Hoving,

Novartis.

Fix the gel at least 3 hours in 50% ethanol / 3%

phosphoric acid (1.5 L for ten gels: 750 mL etha-

nol, 45 mL of 85% H

)

Wash 3  20 minutes in distilled water

Pre-incubate for 1 hour in 34% (v/v) methanol /

3% (w/v) phosphoric acid / 17% (w/v) ammo-

nium sulfate solution:

– First dissolve the ammonium sulfate in water/

phosphoric acid.

– Then add slowly the methanol.

– For 1.5 L (sufficient for ten gels): 45 mL of

85% H

, 860 mL water, 255 g ammonium

sulfate; then add 510 mL methanol.

Gel can stay in stain or fixation

solution over a weekend

without any effect on the

results.

It is said that sensitivity close to

a good silver staining procedure

can be achieved with repeated

staining.

Anderson NL, Esquer-Blasco R,

Hofmann J-P, Anderson NG.

Electrophoresis 12 (1991)

907–930.

6.1 Visible Stains 363

Add 0.53 g Coomassie brilliant blue G-250 pow-

der per 1.5 L solution and stain for 4–5 days on

an orbital shaker.

Wash the gels a few times in water to remove

background stain.

6.1.3

Hot Coomassie Brilliant Blue Staining

Staining Solution: 0.025% (w/v) Coomassie R-350

Dissolve one PhastGel Blue tablet in 1.6 L of

10% acetic acid.

Staining Procedure

Place the gel in a stainless steel tray.

Heat the solution to 90 C and pour it over the

gel in the tray.

Place the tray on a laboratory shaker for 10 min.

Destaining

On a rocking table in 10% acetic acid for at least

2 h at room temperature.

Change solution several times; recycle it by pour-

ing it through a filter filled with activated char-

coal.

Staining and destaining solutions can be used repeatedly.

The gels can now be scanned and evaluated. The selected spots can

be cut out with a spot picker.

6.1.4

Silver Staining

6.1.4.1 Quality of Reagents

Water

For silver staining the water quality must be adequate. Some-

times it is thought, that MilliQ quality is a guarantee for good results.

It happens, however, frequently, that the cartridges are not exchanged

for a long period of time.

There is a simple test: Mix a few droplets of the water with a few

droplets of the silver nitrate solution (from a commercial silver stain-

ing kit or laboratory-made). When the water contains chloride, the

mixture becomes immediately turbid: This water cannot be used.

After 1 day the spots are visible,

the end-point is reached after

4–5 days

Placing a paper towel into the

destaining solution adsorbs the

Coomassie dye.

Sometimes, deionized water is

better for silver staining than

MilliQ water.

In case of emergency: buy deio-

nized water from the super-

market.

Step 6: Staining of Gels364

Ethanol If pure ethanol is not available or too expensive, there are

two possibilities:

Buy a commercial 40% stock solution in a super-

market. For fixing just add the acetic acid.

Use MEK denatured ethanol (methylethyl-

ketone).

Other reagents The reagent quality is very important for good

results. The quality of silver nitrate and formaldehyde is most critical.

The following modification of the silver nitrate technique,

described in Table 6.1, is based on the method by Heukeshoven and

Dernick (1986), and is probably the most sensitive and reproducible

one, when the quality of the chemicals and the water is high, and

when the timing of the steps is exactly kept. The sensitivity of this

method is of approximately 0.05–0.1 ng/mm

If only a few gels have to be stained, an automated stainer (see Fig-

ure 6.1) is very useful. Automatic staining is more reproducible than

manual staining.

Fig. 6.1: Automated gel stainer.

Also known under the name

Vodka.

Do not use otherwise denatured

ethanol, do not use methanol.

Never store the 37% formalde-

hyde in the refrigerator or in the

cold room.

Heukeshoven J, Dernick R.

Electrophoresis. 6 (1985)

103–112.

6.1 Visible Stains 365

Tab. 6.1: Silver staining according to Heukeshoven and Dernick (1986).

Step Solution Volume (mL) Time for gel type

Manual or

in automated

stainer

Laboratory-made solutions or silver

staining kit for proteins

0.5 mm backed or

1 mm unbacked

(min)

1 mm on film or

glass support or

1.5 mm unbacked

(min)

Fixing 10% acetic acid;

40% ethanol;

with deionized water to

2 250 2 15 2 60

Sensitizing 75 mL ethanol;

10 mL Na-thiosulfate (5% w/v);

17 g Na-acetate;

1.25 mL glutardialdehyde;

with deionized water to

250 30 60

Washing Deionized water 3 to 5 250 3 15 5 8

Silver 25 mL silver nitrate ( 2.5%);

100 mL formaldehyde;

with deionized water to

250 20 60

Washing Deionized water 2 to 4 250 2 1 4 1

Developing 6.25 g Na-carbonate;

100 mL formaldehyde;

7 mL Na-thiosulfate (5% w/v);

with deionized water to

250 4 6

Stopping 3.65 g EDTA;

with deionized water to

250 10 40

Washing Deionized water 2 to 3 250 3 5 2 30

The addition of a tiny amount of Na-thiosulfate is particularly help-

ful to reduce the background when thick gels and film-supported

1 mm thick gels are stained.

& Note: When silver stained spots should be

further analyzed with mass spectrometry, some

modifications of this protocol have to be made:

No glutardialdehyde in the sensitizer and no

formaldehyde in the silver solution;

No Na-thiosulfate in the developer.

The staining tray must be covered with a lid

to prevent contamination with keratin.

Step 6: Staining of Gels366

6.1.4.2 Staining of Multiple Gels

Up to four gels can be stained at a time with a special set of staining

trays on an orbital shaker, as shown in Figure 6.2. One tray is perfo-

rated to allow easy changing of staining liquids without breaking the

gels. 1.5 L solution is needed for each step.

Fig. 6.2: Staining of multiple large format gels on an

orbital shaker. Up to four gels, with or without film-backing

can be stained together in one set.

6.1.4.3 Double Staining

By staining gels sequentially in Coomassie blue and silver stain, the

sensitivity of detection can be increased. However, the background of

the Coomassie blue stain must be completely clear. Because the spots

are fixed and the buffer components are already removed, the fixing

steps can be omitted, and the procedure starts with the sensitizing

step.

6.1.5

Scanner Settings for Visible Dyes

Activate in the Scanner Options: “Always use

TWAIN controls.”

Press “Scan”, set the scanning parameters in the

submenu in the Scanner Control Window:

– If necessary, choose light source color;

For instance: Yellow-brown

silver stained spots are best

scanned with blue color.

6.1 Visible Stains 367

– Transmission mode;

– Gray scale;

– Bits per pixel as high as possible, e.g. 16 bits;

– Resolution 300 dpi or lower;

– Gamma correction.

If the value is set above “1”, the intensity resolution will be higher for

the strong signals, if it is set below “1”; the weaker signals are dis-

played with a higher intensity resolution.

These parameters are saved under “Settings”.

6.1.5.1 Calibration of the Scanner

At first the measured dimensionless intensity has to be converted

into optical density (O.D.) values by calibration of the scanner with a

gray step tablet.

Choose “Recalibrate” in the program.

Set the units, usually: “Optical Density”

Scan a gray step tablet, available for instance

from Kodak, shown in Figure 6.3.

Fig. 6.3: Calibration of the scanner with the help of a gray step tablet.

Otherwise you cannot perform

proper quantification.

Important for quantification.

To limit the size of the resulting

file.

A tool for intensity adjustment

at selected level.

Note: It is much easier to cali-

brate the scanner than cali-

brating each gel after scanning.

Step 6: Staining of Gels368

Enter the step wedge intensities into the step

wedge list.

Identify the step wedge points, when a value is

used, it is indicated in the image window. The

software will produce a string of points, which

can be stretched to fit the steps.

Select “Cubic spline”. This curve will be saved as

the default calibration, which can be loaded for

each scan.

6.2

Fluorescent stains

6.2.1

SYPRO Ruby



or RuBPS Staining

For highest sensitivity with Sypro Ruby or RuBPS the following pro-

cedure is recommended by Rabilloud et al. (2001).

The volumes of the staining solutions are optimized for a single

1 mm thick Ettan Dalt gel, which has a volume of approximately

50 mL. For 1.5 mm thick gels the volumes should be multiplied by

1.5. The backing must be nonfluorescent glass or film.

Tab. 6.2: SYPRO Ruby



/RuBPS staining.

Step Solution Volume (mL) Time for gel type

In dark covered

plastic tray

Freshly made 1 mm unbacked 1 mm on film or

glass support or

1.5 mm unbacked

Fixation 20% (v/v) ethanol,

7% (v/v) acetic acid in deionized water

500 1 hour 2 hours

Washing 20% (v/v) ethanol 500 4 30 minutes 4 45 minutes

Staining SYPRO Ruby or RuBPS 700 Overnight Overnight

Washing Deionized water 500 2 10 minutes 2 30 minutes

Stain and store the gels in dark plastic containers. Do not use steel

or glass trays.

Rabilloud T, Strub J-M, Luche

S, van Dorsselaer A, Lunardi J.

Proteomics 1 (2001) 699–704.

6.2 Fluorescent stains 369

Tab. 6.3: Laser and emission filter selection for scanning with

Typhoon.

Dye Laser Emission filter (nm)

SYPRO Ruby or RuBPS Green 532 nm 620

6.2.2

Deep Purple Staining

The volumes of the staining solutions are optimized for one 1 mm

thick Ettan Dalt gel, which has a volume of approximately 50 mL. For

1.5 mm thick gels the volumes should be multiplied 1.5. The back-

ing must be non fluorescent glass or film.

Tab. 6.4: Deep Purple staining.

Step Solution Volume (mL) Time for gel type

In dark

covered

plastic tray

Freshly made [mL] 1 mm unbacked 1 mm on film or

glass support or

1.5 mm unbacked

Fixation 15% (v/v) ethanol,

1% (w/v) acetic acid

(approx. pH 2.3) in deionized water

1,000 1 hour or overnight 2 hours

Staining 0.5% (v/v) Deep Purple in

100 mmol/L sodium borate,

pH 10.5–10.8 in deionized water *)

700 1–4 hours **) 1.5–4 hours **)

Washing 15% (v/v) ethanol in

deionized water

700 30 minutes 45 minutes

Acidification 15% (v/v) ethanol,

1% (w/v) acetic acid (approx. pH 2.3)

in deionized water

250 30 minutes Repeat or extend up

to overnight

Storage Recycled staining solution,

pH adjusted to 2.4 by addition

of 5% (w/v) citric acid and filtered

700

*) Dissolve 6.2g boric acid in 800 mL deionized water and adjust pH to 10.5

with NaOH [approx. 5 mL of a 50% (w/v) solution], then make to 1 L.

The volume of staining solution should be 10–20 times that of the gel.

**) Extending the staining time up to 4 hours does not adversely affect

results. There is some loss in fluorescence intensity if the staining time

is greater than 5 hours.

Step 6: Staining of Gels370

Stain and store the gels in dark plastic containers at 4 C in the sto-

rage solution. Do not use steel or glass trays.

Prior to imaging rinse the gels 2 15 minutes in washing solution.

Tab. 6.5: Laser and emission filter selection for scanning with

Typhoon.

Dye Laser Emission filter

Deep Purple Green 532 nm 580/30 nm

For scanning Deep Purple-stained DIGE gels it is better to scan

with a blue (457 nm) laser with a red (610 nm) band pass filter to

avoid cross-talk.

Tab. 6.6: Excitation and emission filter selection for scanning with EDI

scanning CCD camera imager.

Dye Excitation filter Emission filter

Deep Purple 540/25nm Green 595/25 nm Orange

For scanning Deep Purple-stained DIGE gels it is better to use the

violet excitation filter (390/20 nm) with the orange emission filter

(595/25 nm) to avoid cross-talk.

6.3

Preserving and Drying of Gels

Film-backed gels can be stored in a sealed sheet protector. The open

side is simply closed by sealing it with a kitchen foil welding appara-

tus.

Gels without film support can be dried between two sheets of cello-

phane, which are clamped into specially designed frames, as shown

in Figure 6.4. The cellophane must be prewetted with 10% glycerol /

water without alcohol.

During staining gels tend to swell. When a gel does not fit into

such a frame anymore, it has to be re-shrunk to its original size with

30% ethanol / 10% glycerol.

6.3 Preserving and Drying of Gels 371

Fig. 6.4: Drying of a polyacrylamide gel between two sheets

of cellophane using two plastic frames. A. From the bottom:

Loading platform – smaller frame – pre-wetted cellophane –

gel – pre-wetted cellophane – larger frame. B. The two clamped

frames with the sandwich are removed from the platform; and

the screws are turned by 90 degrees.

This drying procedure can also be applied on film-supported gels.