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

Подождите немного. Документ загружается.

Step 4: SDS Polyacrylamide Gel Electrophoresis352

Fast runs:

Step 1 5 mA per gel »0.2 W 2 hours

Step 2 40 mA per gel 15 W 4–6 hours

In a warm laboratory the buffer temperature can rise to 30 C. This

does not create a problem. When the temperature gets higher, the

power setting should be reduced.

& Note: The running time is also dependent on the

water quality. Water with more ions causes

slower runs.

After the run

Switch off the electric field.

Open the safety lid.

Remove the cassettes.

For DIGE DIGE gels are scanned while they are in the low fluores-

cent glass cassettes. If they cannot be scanned within the next couple

of hours, place the cassettes into a refrigerator or into a cold room.

Do not fix the gels, because acidic fixation would

reduce the fluorescent signal considerably.

For staining the gels

Open the cassettes only with a piece of plastic,

like a Wonderwedge.

Remove the gels from the cassette; be very care-

ful, when gels are not attached to a glass plate or

a support film. Place the gels immediately into

fixing solution or the hot Coomassie blue solu-

tion (see next chapter).

Rinse the cassettes with mild detergent, tap

water and deionized water and let them dry in

the air.

Pump the used buffer out by opening the valve:

Be sure that the end of the tubing is placed into

a bucket or a sink. Switch the pump off immedi-

ately when the tank is empty.

When the anodal buffer should be re-used (only possible for Tris-Cl

gels), pump only 2.5 L out. When the PPA-Cl gels are run, the buffer

cannot be re-used. The Tris ions have to be completely removed from

the lower buffer tank by filling several times a few liters of water in

and pumping it out.

If you use metal spatulas or

knives, you can damage the

glass plates.

Or dry them with lint-free

tissue paper.

Do not walk away during this

procedure!

4.5 The SDS Electrophoresis Run 353

Rinse the cathode panels on the hinged safety

lid with deionized water.

Wash the lower buffer tank several times with

tap water and then with deionized water to

remove any disturbing ions.

4.5.2

The Six-gel Instrument with Standard Power Supply

& Note: Never plug the pump cable in without

liquid in the tank.

Check whether the instrument has been pre-

pared as described above in Section 4.3.3.

Wet the buffer seal of the upper buffer chamber

with 0.1% SDS water.

Insert the cassettes into the cassette carrier and

place it into the tank.

When fewer than six gels are run, insert blank

cassettes into the free positions. In the front,

however, a gel cassette should be inserted: this

makes it easier to watch the migration of the

Bromophenol blue front during the run.

When all cassettes are in place, the level of the anodal buffer should

have reached the mark “LBC start fill”. If necessary, add anodal buffer

or deionized water (for PPA buffer system for ready-made gels) to

reach the mark. The filling procedure is shown in detail in Figure

4.13.

Make up 1.2 L cathodal buffer by diluting and

mixing the concentrate:

– 1 cathodal buffer for running 1–3 gels (120

mL concentrate plus 1,080 mL deionized

water).

– 2 cathodal buffer for more than 3 gels, to pre-

vent buffer depletion (240 mL concentrate

plus 960 mL deionized water).

When a pump runs dry, it gets

destroyed.

Spray with the plant sprayer

used for overlaying the gel

edges.

Turn ready-made gel cassettes

with the glass plates to the

front, because the plastic

frames are not transparent.

Step 4: SDS Polyacrylamide Gel Electrophoresis354

Fig. 4.13: Schematic drawing of the filling steps for the six-gel

chamber. It is important to have a hydrostatic balance between

upper and lower buffer.

Pour 1.2 L cathodal buffer into the upper tank.

Immediately fill more water or buffer into the

lower buffer chamber to adjust the liquid level to

the level in the upper buffer. The hydrostatic bal-

ance is necessary to prevent leakage of the upper

buffer tank and mixing of the buffers (see Figure

4.13).

Put the safety lid containing the cathodal electro-

des in place and connect the cables to the power

supply.

The running conditions are the same for both ready-made gels and

laboratory-made SDS gels:

Overnight runs:

Step 1 5 mA per gel »0.2 W 2 hours

Step 2 25 mA per gel 1 W Overnight

Fast runs:

Step 1 5 mA per gel »0.2 W 2 hours

Step 2 40 mA per gel 15 W 4–6 hours

Water is for ready-made gels /

buffer, for other gels buffer is

poured in.

External thermostat is set to

25 C.

4.6 The SDS Electrophoresis Run 355

& Note: The running time is also dependent on the

water quality. Water with more ions causes

slower runs.

After the run

Switch off the power supply and unplug the con-

necting cables.

Unplug the pump cable.

Remove the safety lid.

Remove the entire cassette carrier and carry it to

a sink. Then remove the upper buffer tank and

the cassettes from the carrier.

When the anodal buffer is required for re-use (only possible for Tris-

Cl gels), the upper buffer must be collected and poured into the ano-

dal buffer in order to bring the Tris ions back.

For DIGE DIGE gels are scanned while they are in the low fluores-

cent glass cassettes. If they cannot be scanned within the next couple

of hours, place the cassettes into a refrigerator or into a cold room.

Do not fix the gels, because acidic fixation would

reduce the fluorescent signal considerably.

For staining the gels

Open the cassettes only with a piece of plastic,

like a Wonderwedge.

Remove the gels from the cassette; be very care-

ful, when gels are not attached to a glass plate or

a support film. Place the gels immediately into

fixing solution or the hot Coomassie blue solu-

tion (see next Chapter).

Clean the glass plates as soon as possible (see

below)!

Pour the buffer out and wash the lower buffer

tank several times with tap water and then with

deionized water to remove any disturbing ions.

When the PPA-Cl gels are run, the buffer cannot be re-used. The Tris

ions have to be completely removed from the lower buffer tank by fill-

ing several times a few liters of water in and pumping it out.

Rinse the cathode panels on the safety lid with

deionized water.

Fixing of gels causes quantita-

tively and qualitatively modi-

fied results.

If you use metal spatulas or

knives, you can damage the

glass plates.

Step 4: SDS Polyacrylamide Gel Electrophoresis356

4.6

Cleaning the Glass Plates

Normal cleaning

Rinse the cassettes with 0.5% (w/v) SDS solu-

tion.

Wash them with tap water.

Rinse them with deionized water.

Let them dry in the air.

Removal of Bind-Silane bound gels from glass plates

Soak the glass plates overnight in a 5% (v/v)

Decon 90 solution.

Remove any gel fragments attached to the plate

from previous gels.

If Decon is not available

Pour a few mL of 0.5 mol/L KOH on the glass

plate, distribute the solution evenly over the sur-

face.

Wait for a few minutes, wipe the gel off with a

paper.

Wash with tap water.

Rinse with deionized water.

Store the glass plates and cassettes in a dust-free

cabinet.

Or dry them with lint-free

tissue paper.

Longer time can cause etching

of the surface.

357

Step 5: Scanning of Gels Containing Pre-labeled

Proteins

Gels containing fluorescence pre-labeled proteins can be scanned

while they are inside the cassette. It is important, that cassettes are made

from low-fluorescent glass plates. Two types of imagers can be used:

Laser scanners with photomultiplier tubes;

White light sources with excitation filters and a

scanning CCD camera.

The key point is the prevention of crosstalk between the different dye

channels. Therefore the quality of the narrow band pass emission fil-

ters is of high importance.

For correct positioning the cassettes and avoiding the development

of Newton’s rings, the way of cassettes placement is very important.

In the Typhoon laser scanner the cassettes are placed on an assembly

of alignment guides, which provide an opening of 200 mm between

the platen and the cassette glass plate to prevent the formation of

Newton rings. The position of the cassettes is easily recognized by

the software as a pre-defined tray area. For the scanning CCD camera

special carrier cassettes are provided, which have an open bottom for

this application.

2-D gels are always scanned in a defined – and the same – orienta-

tion, in order to evaluate and display the image with the low isoelec-

tric points on the left hand side and the low molecular weights pro-

teins at the bottom. The print on the IPG strips is very useful to find

the correct orientation.

The standard resolution for adequate evaluation with DeCyder soft-

ware is a pixel size of 100 mm. The scanner computer should contain

a software package ImageQuant to overlay the different images for

visual control of the separation results and cropping the images.

Wipe off fluorescent contaminations on the

scanner glass platen and glass plates with lint-

free tissue paper soaked in 10 % (v/v) H

and

rinse with double-distilled water.

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

According to a Cartesian coor-

dinate system.

Step 5: Scanning of Gels Containing Pre-labeled Proteins358

5.1

Selection of Imager Settings

5.1.1

Laser Scanner

For excitation the appropriate laser is selected together with the

related emission filter as shown in Figure 5.1 and Table 5.1.

Fig. 5.1: Graph of CyDyes, excitation, and emission filter

wavelengths for scanning CyDyes with Typhoon.

Tab. 5.1: Laser and emission filter selection for scanning DIGE gels

with Typhoon.

Dye Laser Emission filter (nm)

Cy2 Blue 488 nm 520/40

Cy3 Green 532 nm 580/30

Cy5 Red 633 nm 670/30

Adjust the photomultiplier (PMT) voltage for

each wavelength after pre-scanning the entire

gel with a low resolution of 1,000 mm. The lowest

pixel should have at least ten counts. Saturation

must be avoided. The values are usually between

470 V and 550 V.

Select focal plane (+3 mm) for scanning in

between the glass plates.

Saturated spots (>100,000

counts) are indicated in red

color.

This is a specialty of confocal

optics.

5.2 After Scanning 359

5.1.2

Scanning CCD Camera

For the white light source imager the excitation and emission filters

have to be selected as shown in Figure 5.2 and Table 5.2.

Fig. 5.2: Graph of CyDyes, excitation, and emission filter wavelengths

for scanning CyDyes with EDI scanning CCD camera imager.

Tab. 5.2: Excitation and emission filter selection for scanning DIGE

gels with EDI scanning CCD camera imager.

Dye Excitation filter (nm) Emission filter (nm)

Cy2 480/30 530/40

Cy3 540/25 595/25

Cy5 635/30 680/30

For the CCD camera adjust the exposure time

settings for each wavelength (between 0.15 sec-

onds and 4.0 seconds) after a pre-scan of a small

scanning area.

5.2

After Scanning

The files are saved as 16 bit TIFF files. The software saves the differ-

ent channels in two formats: *.ds and *.gel, to which it adds automa-

tically the suffix Cy2, Cy3, or Cy5, and for the selected internal stan-

dard channel it also adds the suffix STANDARD. The *.ds files allow

the scanned images to be overlaid in ImageQuant.

Usually settings are 0.9 seconds

or below to keep scanning time

down. Weaker signals need

longer exposure.

Step 5: Scanning of Gels Containing Pre-labeled Proteins360

If the cassettes have been scanned in the wrong orientation, this

can be corrected with the scanner software, which includes tools for

rotating and flipping images.

361

Step 6: Staining of Gels

Only a selection of staining techniques will be described, those which

are most useful for fast results, good sensitivity, image analysis, quan-

tification, and mass spectrometry compatibility.

6.1

Visible Stains

6.1.1

Sensitive Stepwise Coomassie Brilliant Blue Colloidal Staining

According to Neuhoff et al. (1988).

6.1.1.1 Solutions

Stock staining solution A:

10% (w/v) ammonium sulfate, 2% (w/v) phos-

phoric acid in 100 mL deionized water.

Stock staining solution B:

5% (w/v) Coomassie brilliant blue G-250 in de-

ionized water.

Staining solution:

Mix 2 mL of stock solution B with 80 ml of stock

solution A. Add 20 mL of methanol into the AB

mix.

& Note: The staining solution should never be

filtered because the colloidal dye particles

formed are retained on the filter.

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

Neuhoff V, Arold N, Taube D

and Ehrhardt W. Electrophor-

esis 9 (1988) 255-262.