for notes

Ettan™ DIGE Basic Course

From Sample Labeling to Scanning

© GE Healthcare Europe GmbH, Oskar-Schlemmer-Str. 11, D-80807 München

11/19/2007 1
 for notes
The buffer and solution recipes are at the end of the manual.
The time schedule is for a three-day workshop. Day 1: 09:00

Always wear disposable gloves.

Pre-rehydration of the IPG strips

DeStreak™solution is used as the rehydration solution for later cup loading.
It is advised to use 18 cm long IPG strips for a course, because they are markedly easier to
handle than 24 cm IPG strips.

At least 6 hours before sample application:

 Add 30 µL IPGbuffer 3-11 NL or Pharmalytes 3-10 to 6 mL DeStreak™ solution.
This means: Add 15 µL to each vial DeStreak™ solution.
 Level the reswelling tray horizontal on the bench.

adjust
level IPG strip
rehydration
solution
gel side
down

protection
cover

if necessary remove
airbubbles
paraffin
oil

 For 18 cm IPG strips 340 µL pipette of DeStreak™ solution into each groove of the
reswelling tray as a streak (for 24 cm IPG strips: 450 µL).
 Remove cover film from the IPG strip beginning at the acidic end.
 Place the IPGstrip – gel surface down – on the DeStreak™ solution avoiding air
bubbles.
If necessary, remove air bubbles by lifting the IPGstrip carefully up using a forceps with
bent tips and lower it carefully down again.
 Check, whether the serial number of the strip can be read correctly. If the number
is mirror-converted, the gel surface is turned upside. (which is wrong)
 Pipette 3 mL DryStrip cover fluid onto the IPG strip starting from the ends moving
towards the center.
 Continue with the next IPG strip (follow this sequence for each IPG strip, do not pre-
pipette the solution for several strips).
 Close the sliding lid and leave it at room temperature for at least 6 hours.
11/19/2007 2
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Casting of Large Format SDS gels

Day 1: 14:30
Optional: Preparing the cassettes for spot picking
Treating glass plates with Bind-Silane
The gel is bound to the glass plate without spacers; this one is treated with Bind-silane.
Note: It is important that glass plates are properly clean.
 Before re-use, place the plates in 5% (v/v) Decon™ 90 solution overnight. Do not
leave plates standing in this solution for a longer time, because this will cause
etching due to the alkali nature of Decon™ 90.
(Alternatively place them in 0.5 M NaOH for 30 min, rinse them in plenty of water)
 Thoroughly wash the plate to be treated. Any gel fragments from previous gels
must be removed. The careful cleaning of the glass plates before casting is
important, to ensure a uniform coating with the Bind-Silane and, to avoid keratin
contamination.
 Thoroughly rinse the plates with MilliQ water to remove the Decon (or the NaOH).
 Dry the plate using a lint-free tissue or leave them to air dry.

Application of internal reference markers

 Apply two self-adhesive internal reference marker stickers on the glass plate as
shown in the figure below (the spot picker camera will find the IR markers quicker).

Bind-Silane working solution:

Ethanol 8 mL
Acetic acid 200 µL
BindSilane 10 µL
MilliQ water 1.8 mL

 Pipette 4 mL of the Bind-Silane solution onto the plate and distribute equally over
the plate with a lint-free tissue. Cover the plate to prevent dust contamination and
leave to air dry on the bench for one hour.
 Polish the plate with a lint-free tissue, moistened with a small amount of MilliQ
water or ethanol.
 Once the glass plates have been Bind-silane treated, the gels should be cast on the
same day.
11/19/2007 3
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Ettan™ DALTtwelve Gel Caster (up to 14 gels)
 Before setting up the caster: Make up 1000 mL monomer solution, degas and pre-
cool it in the refrigerator. Day 1: 14:30
 Freshly prepare ammonium persulfate and displacing solution.

Preparation of the gel caster

Use only the low fluorescent glass plates.
Never forget to place the plastic separator sheets between the cassettes; they would firmly
stick to each other after gel polymerization!

 Tip the caster back, so it rests on the support legs.

 Clean low fluorescent glass plates with a lint-free tissue and MilliQ water.
 Place the cassettes alternating with separator sheets into the caster as shown in
the figure below (start with a separator sheet:, assemble cassettes on the bench
before placing them into the caster box).

 Press strongly on the spacers areas on both sides to ensure, that the spacers and
the glass plates stick well together.
 Insert separator sheets and filling plates to complete the stack up to about 1 mm
below the edge of the caster. Perform a “loose” packing. Overfilling would cause
pressure on the gels during polymerisation, when the solution moves up between
the spacers and the glass plates (by capillary forces!) and starts to get warm.
 Place the foam gasket into the groove of the front plate. Do not apply grease.
Always take the foam gasket out of the front plate after gel casting.
 Turn the first four screws into the bottom holes and place the front plate on the
caster with the bottom slots on the screws. Apply the rest of the screws and tighten
them evenly. Do not use too much force; the sealing gasket should be compressed
evenly to prevent leakage.
 Tip the caster to the front.
 Level the gel caster horizontally.
 Add 4 mL ammonium persulfate solution to the degassed and cooled monomer
solution short before gel casting and mix.
 Pour the gel solution directly into the balance chamber, avoiding air bubbles (see
figure below).

11/19/2007 4
 for notes
monomer displacing
solution solution

 Stop pouring when the level has reached 3 cm below the upper edges of the
casting cassettes.
 Pour the dense displacing solution to fill the V-chamber and the sloped bottom of
the caster. The gel solution level will rise to 1 cm below the cassette edges. A thin
blue layer should be visible at the bottom of the cassettes. This saves you from
cutting off protruding gel pieces at the bottom edge of each cassette. And it
prevents a too strong contraction of the gel during polymerisation, which can
cause a gap between gel and spacer
 Immediately spray 0.1 % SDS solution overlay solution over the cassettes (see
figure below).

0.1 % SDS / water

hydrostatic
equilibrium
plant
sprayer

 About 50 mL monomer solution should be left over. Let it polymerise in a small

beaker before you discard it.
 Close the caster with a cling film (e.g. Saran® wrap)
 Let the gel polymerise overnight at room temperature.

SDS Gel concentration: 12.5 %T, 3 % C

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 for notes
Ettan™ DALTsix Gel Caster (up to 6 gels)
Day 1: 14:30
 Before setting up the caster: Make up 500 mL monomer solution, degas and pre-
cool it in the refrigerator.
 Freshly prepare ammonium persulfate.

Preparation of the gel caster

Use only the low fluorescent glass plates.
Never forget to place the plastic separator sheets between the cassettes; they would firmly
stick to each other after gel polymerization!

 Lay the gel caster flat on the bench. For casting homogeneous gels, leave the V-
shaped rubber insert in place.
 Clean low fluorescent glass plates with a lint-free tissue and MilliQ water.
 Place the cassettes alternating with separator sheets into the caster (start with a
separator sheet:, assemble cassettes on the bench before placing them into the
caster box). See figure on page 4.
 Press strongly on the spacers areas on both sides to ensure, that the spacers and
the glass plates stick well together.
 Insert separator sheets and filling plates to complete the stack up to about 1 mm
below the edge of the caster. Perform a “loose” packing. Overfilling would cause
pressure on the gels during polymerisation, when the solution moves up between
the spacers and the glass plates (by capillary forces!) and starts to get warm.
 Place the foam gasket strip into the groove of the front plate. Do not apply grease.
Always take the foam gasket out of the front plate after gel casting.
 Turn the two screws into the bottom holes and place the front plate on the caster
with the bottom slots on the screws. Apply the six clamps and tighten the screws
evenly. Do not use too much force; the sealing gasket should be compressed
evenly to prevent leakage.
 For casting, place the gel caster upright in a tray, for the occasional case that liquid
is overflowing.
 Close the filler port at the bottom of the front plate with the cap or with a piece of
tubing and a pinchcock clamp.
 Add 2 mL ammonium persulfate solution to the degassed and cooled monomer
solution and mix.
 Pour the gel solution directly into the box, avoiding air bubbles (see figure below).

monomer
solution 0.1 % SDS / water

plant
sprayer

11/19/2007 6
 for notes
Stop pouring when the level has reached 3 cm below the upper edges of the casting
cassettes.
 Immediately spray 0.1 % SDS solution overlay solution over the cassettes.
 Cover the caster with a cling film (Saran® wrap).
 Let the gel polymerise overnight at room temperature.

SDS Gel concentration: 12.5 %T, 3 % C

11/19/2007 7
 for notes

Planning of the Experiments

Perform a “planned randomization” of Dye labeling and application on IPG strip. Day 1: 15:45

Sample / Cy3 Conc Sample / Cy5 Conc Int. stand. / Cy2 IPG strip Grad.
(µL of 50 µg) (µL of 50 µg) no. cm
1

10

11

12

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 for notes

Sample Preparation
 Add DIGE “Lysis”-Buffer (labeling buffer) to each sample to a concentration in the
range Day 1: 15:45

5-10 mg/mL
Independently from the experiment size, at least 75 µg of each sample is required: 50 µg
for sample labeling, 25 µg for creating the internal standard.
If accurate quantification of the samples´ protein amount could not be determined: under
labeling is not an issue. Only over labeling would create multiple labels in the basic area.

Quantification
It is recommended to use the Ettan™ 2-D Quant Kit

Clean up
It is recommended to use the Ettan™ 2-D Clean-Up Kit

pH control and adjustment

For efficient labeling the protein sample should have optimally pH 8.5; it must be above
pH 8.0.
 Check the pH carefully: pipette 2 µL sample on a pH indicator paper. Read out the
pH value immediately, because the color will shift with time.
If necessary, adjust the pH value with adding 100 mM NaOH solution.

Note: Proteins have some inherent buffering capacity and may have decreased the pH
value of the sample solution below pH 8. Samples which have been cleaned up with TCA
acetone or the Ettan™ 2-D cleanup kit can be acidic. Beware: Sometimes, when samples
have been transported in dry ice and the tubes have not been sealed well enough, CO2 has
diffused into the samples and caused a strong drop of the pH value. In this case it might be
necessary to adjust the pH value with 250 mM NaOH.

Pooling of the Internal Standard

For each experiment:
 Take an aliquot of 25 µg from each sample and mix them together in one vial.

11/19/2007 9
 for notes

CyDye Labeling
Day 1: 15:45
Reconstitution of the CyDyes
Use 99.8% anhydrous Dimethylformamide (DMF) less than 3 months old from day of
opening. The quality of the DMF is critical to ensure that the protein labeling is successful.
The DMF must be anhydrous and every effort should be used to ensure it is not
contaminated with water. DMF after opening, over a period of time, will degrade with
amine compounds being produced. Amines will react with the NHS ester CyDye reducing
the concentration of dye available for protein labelling. Adding a 4 Å molecular sieve to
DMF during storage is a good measure to prolong the useful lifetime of DMF.

Stock dye solutions

Reconstitute CyDye minimal dyes solid compounds DMF to a concentration of:
1 nmol/µL
e.g. 5 µL DMF to 5 nmol/L of dye.
The stock solution of Cy2 will have a deep yellow, Cy3 a deep red, and Cy5 a deep blue
color.
The solutions are stable at –20 °C for several months.
 Take a small volume of DMF from its original container and dispense into a
microcentrifuge tube.
 Take the CyDye from the –20 ºC freezer and leave to warm for 5 minutes at room
temperature.
 After 5 minutes: add 5 µL of the DMF to each new vial of CyDye.
 Replace the cap on the dye microcentrifuge tube and vortex vigorously for 30
seconds.
 Centrifuge the microcentrifuge
tube for 30 seconds at 12,000 g
in a benchtop microcentrifuge. CyDye DMF
The dye can now be used. Stock solution 5 nmol + 5µL
stable for 3 months
Working dye solutions →1000 pmol / µL
 Briefly spin down dye stock
solution in a microcentrifuge dye DMF
 Dilute 1 volume of the stock 2 parts + 3 parts
Working solution
CyDye in 1.5 x volumes of high 5 µL 7.5 µL
grade DMF to create: →400 pmol / µL
400 pmol/µL
 Add 3 µL of the DMF first to the Labeling 1 µL dye protein
sterile microcentrifuge tube. mixture
 Add 2 µL of the stock dye and mix. 400 pmol + 50 µg

Now you have 2,000 pmoles CyDye

lysine
Quench 1 µL 10 mmol/L
in 5 µL

11/19/2007 10
 for notes
Shortcut to working dye solution
Take a small volume of DMF from its original container and dispense into a
microcentrifuge tube. Take the CyDye from the –20 ºC freezer and leave to warm for 5
minutes at room temperature.
 After 5 minutes: add 12.5 µL of the DMF to each new vial of CyDye. Replace the cap
on the dye microcentrifuge tube and vortex vigorously for 30 seconds.
 Centrifuge the microcentrifuge tube for 30 seconds at 12,000 g in a benchtop
microcentrifuge.
The dye can now be used.

Note: CyDye in the diluted form is only stable for 2 weeks at –20ºC.

Labeling of the samples and the internal standard

Samples:
 Add a volume of sample equivalent to 50 µg protein to a microcentrifuge tube.
 Add 1 µL of diluted CyDye to the microcentrifuge tube containing the sample.
 Mix and centrifuge briefly in a microcentrifuge. Leave on ice for 30 minutes in the
dark.
 Add 1 µL of 10 mmol/L lysine to stop the reaction. Mix and spin briefly in a
microcentrifuge. Leave for 10 minutes on ice in the dark.

Internal standard
n is the number of gels in the experiment
 Add a volume of pooled internal standard equivalent to n × 50 µg protein to a
microcentrifuge tube.
 Add n µL of diluted Cy2 to the microcentrifuge tube containing the pooled standard
(i.e. 300 µg of protein would be labeled with 2,400 pmoles of dye).
 Mix and centrifuge briefly in a microcentrifuge. Leave on ice for 30 minutes in the
dark.
 Add n µL of 10 mmol/L lysine to stop the reaction. Mix and spin briefly in a
microcentrifuge. Leave for 10 minutes on ice in the dark.

Labeling is now finished:

400 pmol per 50 µg protein
Samples and pooled standard can now be stored for at least three months at −70°C in
the dark.

Preparation for loading the samples onto the IPG strips

 Combine the labeled samples and pooled internal standards according to the
experimental design.
 Add an equal volume of 2 x lysis buffer (containing the IPG buffer and the DTT) to
each sample and standard, and leave on ice for 10 minutes.
 If necessary, dilute samples further with a 1:1 mix of DIGE “Lysis”-Buffer (labeling
buffer) and 2 x lysis buffer to a minimum of 100 µL for optimum protein entry.
In highly concentrated samples some proteins tend to aggregate and precipitate.

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 for notes

Isoelectric Focusing Day 1: 16:45

The IPGphor is connected to an external computer via the serial port to control and
monitor the electrical conditions. This allows to judge from the shape of the graphs,
whether the separation will give good or bad 2-D results.
 Level the IPGphor chamber must be horizontally on the bench.
 Be sure, that the manifold is carefully cleaned and dried.
Never use new strip holders without cleaning them before the first run.
 Place the Manifold on the cooled electrode contact areas of the power supply.
 Starting at the basic side, place the IPG strip – gel side facing up – into the strip
holder with the acidic end towards the anode side. Be sure that the protruding film
at the basic end touches the end of the groove. Check: Now the number printed on
the strip must be mirror-converted, the gel surface has to be turned upside.
Aligner protrusions along the grooves inside the manifold align the rehydrated IPG strips,
keeping them straight and centered when placed inside the manifold.
 Soak electrode pads with Milli-Q water.
 Blot them on filter paper and place them on top of the ends of the strip.
The pads should sit completely on the gel surface. If longer pads are required for removal
of salt, there must be an overlapping of at least 5 mm. The pads must be damp, not wet.
The electrode assembly has electrode teeth on one side and hold-down teeth (for
paperbridge-loading) on the other side. It is important to choose the correct orientation,
to get contact with the electrode pads.
 Place the electrode assemblies on the pads. Secure them on place with the cams.
 Apply the loading cups at correct side of the strip. Press them down with the
“sample cup insertion tool” to prevent leakage.
Mostly anodal sample application is employed.
Note: The cup can straddle on the alignment protrusions, if necessary
 Pour 100 mL Drystrip cover fluid (paraffin oil) over the strips, around the cups.
Any leakage would be detected, because oil would flow into a cup.
electrode assembly
gel
acidic
surface up Sequence:
end
+
pads
soaked
1. apply strips
in water
2. apply pads

3. apply electrodes
hold down
teeth
4. apply cups
electrode
teeth 5. press cups down

6. pour oil around cups

loading cups

sample
(check cups for leakage)
paraffin
oil
7. apply samples

8. apply oil on samples

9. close lid

 Pipette samples into the cups.

 Pipette 20 µL paraffin oil on each sample.
 Close the safety lid.

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 for notes
Set up running conditions
 Enter the running conditions in the computer.
 Start Ettan™ IPGphor3 program.
 Select the instrument connected (usually instrument 1 of four)
 Select pI range, strip length and number of strips. A programmed voltage running
curve will show up.
 Click on the “table” icon on the right low position of the screen (red arrow).

 Enter the running parameters as shown in the table below.

Rehydration time 0h

Temperature 20 °C
Current per strip 75 µA
Strip length 18 cm 24 cm 24 cm
pH gradient 3-11 NL 4-7
Step 1 step & hold 150 V 3h
Step 2 step & hold 300 V 3h
Step 3 gradient 1,000 V 6h
Step 4 gradient 10,000 V 1h
Step 5 step & hold 10,000 V 2:00 h 3:00 h 5:00 h
Total time [h] 15:00 16:00 18:00

 Save method under a new name.

 Transfer to IPGphor instrument.

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 for notes
Next day
 Control the focusing advancement in the IPGphor and the voltage / current graphs
Day 2: 09:00
on the laptop computer.

When the overnight run is finished, let the IPGstrips in the IPGphor, with the instrument
switched on until they are needed. The proteins will be refocused with 10,000 V for 15
minutes before equilibration.

Two alternative program variants can be performed:

Preferred procedure: Fast SDS run during Day 2

Start of the SDS run in the morning, the run will be finished in evening. The cassettes are
removed from the electrophoresis chamber and stored in the refrigerator overnight.
When time allows: scan one or two cassettes immediately after the run.

Alternative: Slow SDS run overnight until Day 3

Start of the SDS run in the afternoon, The run will be finished in the morning, followed
directly by scanning the cassettes….

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 for notes

SDS Electrophoresis
 During unloading rinse the cassettes with tap water to remove excess Day 2: 09:00
polyacrylamide.
 Inspect each gel cassette for eventual air bubbles. Gels with air bubbles should not alternatively:
be used. Day 2: 15:45
 Place each cassette into the cassette rack.
 Take the foam gasket out of the front plate and rinse it with deionized water. Do
not leave it in the groove, because it would loose its sealing property.
 Rinse the gel caster and the separator sheet with 0.5 % (w/v) SDS solution and
then with MilliQ water. Let them dry in the air.

Preparing the equilibration solution

The frozen solution aliquoted in 50 mL centrifuge tubes need some time to thaw.
 Take 200 mL of equilibration buffer from the freezer and let them thaw at room
temperature.
 Weigh out 1 g DTT and 2.5 g iodoacetamide.

Refocus the IPGstrips

 Refocus the proteins by applying 10,000 V on the strips for 15 minutes before
equilibration (only necessary when strips left in IPGphor for longer than ½ hour).

Setting up the Ettan™ DALTtwelve

 Place the separation unit on a levelled bench. Check with a spirit level. If necessary,
adjust the level with inserting plastic sheets below some feet of the separation unit.
 Switch on the main switch.
 Turn the pump valve at the back of the separation unit to “circulate”.
 Pour 950 mL running buffer (10 x conc) into the lower buffer tank.
 Fill the tank up to the mark 9.5 L with MilliQ water.
 Set the temperature to 25 °C, and set the pump to “ON”. The pump starts to
circulate the liquid, mixes the concentrate with the water, and the buffer
temperature will be adjusted to 25 °C.
 Check, whether the buffer circulates; if needed tilt the separation unit by 20° left
and right to remove the air-pocket in the pump housing.

Setting up the Ettan™ DALTsix modular system

 Take the cassette carrier and the upper buffer chamber out of the instrument.
 Connect the tubing to a circulating thermostat, which has been set to 10 °C.
 Pour 450 mL running buffer (10 x conc.) into the lower buffer tank.
 Fill 4 L MilliQ water into the tank.
 Plug the cable of the pump in. The pump starts to circulate the liquid, mixes the
concentrate with the water.

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 for notes

Equilibration of the IPG strips Day 2: 09:30

 Remove the electrodes, the loading cups, and the electrode pads from the Manifold.
 Pour the dry strip cover fluid out from the Manifold. alternatively:
 Add 1 g DTT to 100 mL equilibration buffer, mix thoroughly and pour into the Day 2: 15:45
Manifold.
 Place the manifold on an orbital shaker for 15 minutes, which is set to 30 rpm.
 After 15 min, pour out the first equilibration buffer.
 Add 2.5 g iodoacetamide to 100 mL equilibration buffer, mix thoroughly and pour
into the Manifold.
 Place the Manifold on an orbital shaker for another 15 minutes, which is set to 30
rpm.
 After 15 min, pour out the second equilibration buffer.
Do not leave the strips longer in equilibration buffer, because this would elute a part of
the proteins from the strip.

Application of the IPG strips onto the SDS gels

Day 2: 16:15
 Heat pre-prepared agarose on a heating stirrer or in microwave oven to melt it.
 Pour a few milliliters of MilliQ water on the upper gel edge using a squeeze bottle.
This will greatly facilitate the insertion of the IPG strip into the cassette. alternatively:
 Lay the cassette on the bench with the longer glass plate down, the protruding Day 2: 10:00
edge oriented towards the operator.
 Place the IPG strip with the acidic end to the left, gel surface up onto the protruding
edge of the longer glass plate as shown in the figure below.
The correct orientation of the IPG strip is particularly important for Spot-picking gels ( one
glass plate treated with Bind-silane)

60
hot
agarose
50

40

30

20

10

gel surface up
acidic end

 With the forceps move the strip into the cassette slot.
 Place the cassette into the rack, with the IPG strip-supporting edge upside.
 With a thin plastic ruler, gently push the IPG strip down so that the entire lower
edge of the IPG strip is in contact with the top surface of the SDS gel (see figure
above). Do not push the IPG strip down too hard, it would force a gap between gel
surface and glass plate and damage the gel.
 Tilt the cassette by 90° to get rid of the water.
 Allow the agarose to cool until the tube can be hold by fingers (60°C) and then
slowly pipette 2 mL agarose solution in to seal the IPG strip in place. Pipetting
slowly avoids introducing bubbles.
The agarose must not be too hot; carbamylation of proteins must be avoided.
11/19/2007 16
 for notes
Inserting the cassettes
Ettan™DALTtwelve
 Wet the tubing of the buffer seal with 0.1 % SDS water. Spray with the plant
sprayer used for overlaying the gel edges.
 Insert the cassettes between the tubing of the buffer seal, starting at the back.
Slide them down to the bottom.
 Do not force the cassettes down; this could damage the buffer seal. Take care, that
the silicone tubing are not bent and stretched down; this would cause current
leakage and buffer mixing. If you detected bent tubing, move the upper edges of
the two neighbouring cassettes slightly back and forward, to release the tubing.
When less than 12 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.

Ettan™DALTsix
 Wet the buffer seal of the upper buffer chamber with 0.1 % SDS water. Spray with
the plant sprayer used for overlaying the gel edges.
 Insert the cassettes into the cassette carrier and place it into the tank.
When less than 6 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 MilliQ water to reach the mark. The filling procedure
is shown in detail in the figure below.
 Make up double concentrated upper buffer by adding 280 mL running buffer (10 x
conc) to 1.12 L MilliQ water. Mix thoroughly.
 Fill the upper buffer chamber with the 1.4 L double concentrated running buffer.
 Immediately fill 1 x concentrated running buffer into the lower buffer tank to the
same level in order to establish a hydrostatic balance.

Running conditions in both instruments

Step 1 10 mA per gel ≈0.5 W /gel 1 hour
Fast run Step 2 15 W/gel ca. 4 hours
Over night run Step 2 25 mA per gel 1.5 W / gel Over night

11/19/2007 17
 for notes
After the SDS run
 Switch on the Typhoon™ scanner and the computer; the scanner needs at least 30
Day 2: 15:00
minutes to warm up. Day 3: 08:30
 Check, whether the Bromophenol Blue front has reached the end of the gels. If not,
increase the power setting to 17 W per gel.
 Stop the SDS electrophoresis separation.
 ED6: remove first the upper buffer chamber, then take the cassette holder out.
 Remove the cassettes from the apparatus, rinse them with tap water, dry them
with lint-free tissue, and place them into the cassette rack.

Scanning the Gels

Typhoon trio 9100

 Put the alignment guides in place on the platen.

 Place cassettes on the alignment guides with the correct orientation as shown
below.

grippers

front location bar

 Close the instrument lid.

Select scan parameters

 Start Typhoon™ Scanner Control software in the computer.
 Select Multiple Sample scan.
 Select Tray-set-up area (1).
 Select scan area (pre-defined tray area)…DIGE Ettan™ DALT
 Define the number of gels.
 Select Acquisition mode …. Fluorescence
 Select Setup.
 Select number of scan channels and Emission Filters. Sensitivity normal.
 Set the PMT voltage to 500 for each channel.
 Orientation (“R”): default (if the cassettes are oriented as shown in the figure above.
 Select “press sample” when gels are inside glass cassettes.
 Select Focal plane +3 mm.
 Set pixel size: 1000 microns for the prescan.

11/19/2007 18
 for notes
Scanning
 Select DIGE File naming format. This format is recognized by the DeCyder™
evaluation software automatically. Use the IPG strip number to name the file. Make
sure, that the Cy2 channel is selected for the standard. The Typhoon™ Scanner
Control software will create a folder with the IPG strip number ******.DIR which
contains the image files with automatically added relevant information: for
instance ****** STANDARD CY2.gel and a file to show the overlaid images ******.ds
 Start the scan by selecting SCAN. The prescan will take about 3 minutes.
 Check the scanning process. Saturated values are indicated in red. In this case
reduce the PMT voltage accordingly…..
 Check the results: Avoid 0 pixels. Avoid saturation.
 Adjust PMT settings that max values for different channels are similar.
Max values should not vary by more than 15 %. If max values are not balanced within
this tolerance, spot co-detection in DeCyder™ might not work properly.
Pixel sizes:
 Large format gels (18 and 24 cm IPG strips) should be scanned with 100 µm.
 Medium formats (11 and 13 cm IPG strips) 50 µm.
 Small format (7 cm IPG strips) 25 µm.

Ettan DIGE Imager

 Turn on the scanner and the computer.
 When READY light is on, double click the Ettan DIGE icon on the desktop to
start the DIGE software.
 Clean the gel glass plates with distilled water using a lint-free tissue. It is important
that the glass plates are clean, dry and free from lint.
 Insert the dried cassette into the EDI cassette with the small plate facing down,
resting in the seal at the bottom of the cassette. The large plate should hang over
the area at the rear of the cassette.
Note: The acidic side of the IPGstrip should point to the left. If the acidic side points to the
right, the image needs to be flipped later on: either in the EDI Report Viewer or later with
ImageQuant.
 Put the lid on the cassette, and close the cassette by turning the locking cams up.
 Slide the Scanner door until it is fully open.
 Insert the cassette into the scanner with the cams towards you and tilted up, so
that the cassette fits under the beveled edge of the carrier. Push it down and in on
the front of the cassette to lock it in place. Close the Scanner door.
 Start the Scanner software by double clicking the Ettan™ DIGE Imager icon on the
computer desktop.

Select scan parameters

 Select the appropriate gel format. The scan area is shown as a blue box in the Grid
Area
 Specify the type of chemistry used for your sample by selecting an item in the
Chemistry list.
 Select the pixels size, i.e. resolution to use, in the Pixel size list. 100 µm are selected
for analysis by DeCyder™.
 Select the number of channels to be scanned by clicking the Channel check box.
Selection of the Channel check boxes must be performed in sequential numerical
order.
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 For each selected channel, select the respective dye. When selecting dye, the
excitation and emission filter are automatically presented by the software. The
excitation and emission filter combinations have been selected to give the
optimum results with minimal crosstalk.
 Select DIGE file naming format, and have a standard defined in your experiment,
specify which dye (usually Cy2) represents the standard under Standard.
 Select the exposure time to be used for each channel in the Exposure time list.
 Perform a quick test scan on a small area to identify a suitable exposure time for
each channel. Therefore select an area containing the most intense spots.
Saturated areas are shown in red.

Example:
Fluorophor Exposure levels (seconds)
Cy2 0.8
Cy3 0.3
Cy5 0.5

CAUTION: The maximum pixel value should not exceed 65,000 counts. If some of the pixel
values are equal to or greater than 65,000 counts, part of the image is at saturation. This
will prevent correct quantitative analysis.
Commonly a target signal of 30,000 to 55,000 is suitable.
 Scan all similar gels within one experiment with the same exposure times like the
gel used for optimization, that max values for different channels are similar.
Max values should not vary by more than 15 %. If max values are not balanced within
this tolerance, spot co-detection in DeCyder™ might not work properly.

Scanning
 Click the Scan button to start the scan.
 In the File Name Dataset dialog box, browse to a directory and enter a name for the
dataset, or choose New folder to create a new folder.
Note: Depending on whether the DIGE File Naming Format check box has been selected
or not the scanned files will end up with different file names and folder structure.
Note: Dataset names must be unique. If you enter the name of an existing dataset, you
will be prompted to overwrite it.
 Click Proceed to start the scan.

Note: When scanning more than one channel, scan data files (in a .gel format) are
created in the dataset directory. Two identical index files (in a .ds format) are also created.
One index file resides in the dataset directory. The other index file resides at the same
level as the dataset directory. When scanning one channel only scan data files in a .gel
format are created.

Cleaning of Typhoon™ platen and EDI cassettes

 Clean platen and cassettes with MilliQ water using a lint free tissue. If required 75%
Ethanol may also be used.
 If fluorescent material has come into direct contact with the platen or cassette use
lint-free tissue moistened with 10% hydrogen peroxide, followed by cleaning with
MilliQ water.

11/19/2007 20
 for notes

Consumables and Recipes for Buffers and Solutions

Consumables

Immobiline DryStrip gels (choice)

DryStrips 3-10NL (12/pke) 18 cm 17-1235-01
DryStrips 3-11NL (12/pke) 18 cm 17-6003-76
DryStrips 4-7 (12/pke) 18 cm 17-1233-01

IPG buffer / Pharmalytes pH 3-11

IPG-Buffer pH 3-11 NL 17-6004-40
Pharmalytes™ pH 3-10 17-0456-01

CyDyeTM DIGE Fluors, minimal dyes

CyDye DIGE Fluor Cy2 minimal dye Cy3 minimal dye Cy5 minimal dye
5 nmoL 25-8010-82 25-8010-83 25-8010-85
Labeling kit, 5 nmoL each 25-8010-65
10 nmoL 25-8008-60 25-8008-61 25-8008-62
25 nmoL 25-1900-27 25-1900-28 25-1900-30

Related Products: EttanTM Reagents and Preparation Kits

Paraffin oil (DryStrip cover fluid) DryStrip Cover Fluid, 1 L 17-1335-01
Dimethyl Formamide (DMF), ultrapure N,N-Dimethyl Formamide, 250 mL 25-0089-85
2-D Clean-Up Kit for 50 samples 80-6484-51
2-D Quant Kit for 500 assays 80-6483-56

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 for notes

Solutions and buffers

Bromophenol Blue Solution

1 % Bromophenol Blue PlusOne™ Bromophenol Blue (17-1329-01) 100 mg
0.06 % Tris PlusOne™ Tris (17-1321-01) 60 mg
to final volume 10 mL
Store at room temperature.

NaOH Solution
100 mmol/L NaOH 1mol/L NaOH solution 1 mL
to final volume 10 mL
Store at room temperature.

DIGE „Lysis“-Buffer (Labeling Buffer)

7 M Urea PlusOne™ Urea (17-1319-01) 21 g
2 M Thiourea PlusOne™ Thiourea (25-9000-65) 7,6 g
4 % CHAPS PlusOne™ CHAPS (17-1314-01) 2g
30 mM Tris PlusOne™ Tris (17-1321-01) 182 mg
MilliQ water to final volume 50 mL
Instead of urea/thiourea 8 M urea (24 g) can be used.
Divide into 5 mL aliquots and store in freezer

DIGE „Stop“-Solution
10 mM Lysine (Mw 182,6) Lysine (Sigma, L-5626) 18 mg
MilliQ water to final volume 10 mL
Divide into 1 mL aliquots and store in freezer

DIGE DeStreakTM „Rehydration“-Solution

Rehydration Solution DeStreak Rehydration Solution (17-6003-19) 3 mL
0,5 % v/v Pharmalyte™ e.g. Pharmalyte™ pH 3-10 (17-0456-01) 15 µL
Prepare fresh

Deionizing the Acrylamide, Bis stock solution short before use

EDtwelve EDsix
Acrylamide, Bis solution PlusOne ReadySol (17-1310-01) 350 mL 175 mL
(40% T, 3% C)
Mixed bed ion Amberlite IRN-150L (17-1326-01) 6g 3g
exchanger
Stir for 10 minutes and filter the solution. Prepare fresh short before use.

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 for notes
DIGE 2x „Lysis“-Buffer
7 M Urea PlusOne™ Urea (17-1319-01) 21 g
2 M Thiourea PlusOne™ Thiourea (25-9000-65) 7.6 g
4 % CHAPS PlusOne™ CHAPS (17-1314-01) 2g
0.04 % Bromophenol Blue Bromophenol Blue solution (1%) 200 µL
to final volume 50 mL
Instead of urea/thiourea 8 M urea (24 g) can be used.
Divide into 5 mL aliquots and store in freezer
Before use add to 5 mL stock:
2 % DTT PlusOne™ Dithiothreitol (17-1318-01) 100 mg
2 % v/v IPG buffer e.g. Pharmalyte™ pH 3-10 (17-0456-01) 100 µL
DIGE 2x „Lysis“-Buffer stock 5 mL
Prepare fresh

SDS Gel Buffer Tris-Cl pH 8.8 (4 x conc)

1.5 mol/L Tris-base PlusOne™ Tris (17-1321-01) 181.8 g
0.4 % SDS PlusOne™ Sodium Dodecylsulfate (17-1313-01) 4g
MilliQ-Water dissolve 800 mL
4 mol/L HCl titrate to pH 8.8
MilliQ-Water to final volume 1L
Store in the refrigerator

Ammonium persulfate solution (APS)

10 % APS PlusOne™ Ammonium Persulfate (17-1311-01) 1g
MilliQ-Water to final volume 10 mL
Prepare fresh

Homogenous monomer solution 12,5% T

EDtwelve EDsix
Acrylamide, Bis solution PlusOne™ ReadySol (17-1310-01) 312 mL 156 mL
(40% T, 3% C) freshly deionized (see page 22)
0.375 M Tris-HCl pH 8,8 SDS Gel buffer Tris-Cl pH 8.8 250 mL 125 mL
TEMED (100%) PlusOne™ TEMED (17-1312-01) 500 µL 250 µL
MilliQ-Water 434 mL 217 mL
10% APS Ammonium Persulfate solution 4 mL 2 mL
total volume 1000 mL 500 mL
Prepare fresh, degas for 15 minutes while stirring, and pre-cool in refrigerator

Displacing solution
0.375 mol/L Tris-Cl pH 8.8 SDS Gel buffer Tris-Cl pH 8.8 30 mL
50 %Glycerol PlusOne™ Glycerol 87 % (17-1325-01) 71 mL
0.02 % Bromophenol Blue 1 % Bromophenol blue solution 240 µL
MilliQ-Water to final volume 120 mL
Prepare fresh

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Overlay spraying solution

0.1 % SDS PlusOne™ Sodium Dodecylsulfate (17-1313-01) 1g
MilliQ-Water to final volume 1L

Gel storage solution (only needed when gels are stored > 1 day)
0.375 mol/L TrisCl pH 8.8 SDS Gel buffer Tris-Cl pH 8.8 500 mL
MilliQ-Water to final volume 2L
Store in the refrigerator

SDS Running Buffer (10 x conc) = „Laemmli buffer“

0.25 mol/L Tris PlusOne™ Tris (17-1321-01) 30.4 g
1.92 mol/L glycine PlusOne™ Glycine(17-1323-01) 144.0 g
1 % (w/v) SDS PlusOne™ Sodium Dodecylsulfate (17-1313-01) 10 g
MilliQ-Water to final volume 1L
Store at room temperature.

Agarose sealing solution:

0.5 % Agarose NA 0.5 g
0.02 % Bromophenol Blue Bromophenol Blue solution (1%) 200 µL
SDS running buffer (10 x conc) 10 mL
MilliQ-Water to final volume 100 mL
Prepare aliquots of 15 mL in centrifuge tubes and store at room temperature.

Equilibration-Buffer
6 M Urea PlusOne™ Urea (17-1319-01) 72 g
2 % SDS PlusOne™ Sodium Dodecylsulfate ((17-1313-01) 4g
50 mM Tris pH 8,8 SDS Gel buffer Tris-Cl pH 8.8 7 mL
0.02 % Bromophenol Blue Bromophenol Blue solution (1%) 400 µL
30 % Glycerol PlusOne™ Glycerol 87 % (17-1325-01) 70 mL
MilliQ-Water to final volume 200 mL
Store in freezer

Equilibration Solution 1 per Manifold (15 min)

100 mL Equilibration-Buffer + 1 g DTT PlusOne™ Dithiothreitol (17-1318-01)
Prepare fresh
Equilibration Solution 2 per Manifold (15 min)
100 mL Equilibration-Buffer + 2.5 g IAA PlusOne™ Iodoacetamide (25-9000-66)
Prepare fresh

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