User Manual - GE Healthcare Life Sciences

User Manual - GE Healthcare Life Sciences
user manual
proteomics
User Manual
um 18-1147-61
Important user information
All users must read this entire manual to fully
understand the safe use of EttanTM Spot Picker.
WARNING!
The Warning sign highlights an instruction
that must be strictly followed in order to avoid
personal injury. Be sure not to proceed until
the instructions are clearly understood and all
stated conditions are met.
Caution!
All goods and services are sold subject to the terms and
conditions of sale of the company within the Amersham
Pharmacia Biotech group which supplies them. A copy
of these terms and conditions is available on request.
Ettan, ImageMaster, ImageScanner, PlusOne, Hoefer,
GelBond, Immobiline, and Typhoon are trademarks of
Amersham Pharmacia Biotech Limited.
Amersham is a trademark of Nycomed Amersham plc.
Pharmacia and Drop Design are trademarks of
Pharmacia & Upjohn Inc.
Gilson is a trademark of Gilson Inc.
The Caution sign is used to call attention to
instructions or conditions that must be
followed to avoid damage to the product or
other equipment. Be sure not to proceed until
the instructions are clearly understood and all
stated conditions are met.
Declaration of conformity
Safety standards
Coomassie is a trademark of Imperial Chemical
Industries PLC.
Decon is a trademark of Decon Laboratories Limited.
SYPRO is a trademark of Molecular Probes Inc.
Microsoft, Microsoft Excel, Microsoft Word and
Windows NT are trademarks of Microsoft Corp.
This product meets the requirements of the Low Voltage
Directive 72/23/EEC through the harmonized standard
EN 61 010-1:1993 + A2:1995.
©Amersham Pharmacia Biotech AB 2000
-All rights reserved
EMC standards
Office Addresses
This product meets the requirements of the EMC
Directive 89/336/EEC through the harmonized
standards EN 50081-1 and EN 50082-1.
Amersham Pharmacia Biotech AB
WARNING!
Amersham Pharmacia Biotech UK Limited
This is a Class A product. In a domestic environment,
this product may cause radio interference, in which case
the user may be required to take adequate measures.
The CE symbol and corresponding declaration of
conformity is valid for the instrument when it is:
–
used as a stand-alone unit, or
–
connected to other CE-marked
Amersham Pharmacia Biotech
instruments, or
–
connected to other products
recommended or described in
this manual, and
–
used in the same state as it was
delivered from Amersham
Pharmacia Biotech except for
alterations described in this
manual.
SE-751 84 Uppsala
Sweden
Amersham Place Little Chalfont
Buckinghamshire
England HP7 9NA
Amersham Pharmacia Biotech Inc.
800 Centennial Avenue
P.O. Box 1327 Piscataway N.J. 08855-1327
USA
Amersham Pharmacia Biotech Europe GmbH
Munzinger Strasse 9
D-79111 Freiburg
Germany
Amersham Pharmacia Biotech KK
Sanken Building
3-25-1 Hyakunincho, Shinjuku-ku
Tokyo
Japan
Contents
1 Introduction
1.1
1.2
1.3
1.4
1.5
The Ettan Spot Picker system ..................................................... 11
Method overview ........................................................................ 12
The User Manual ....................................................................... 13
Accessories ............................................................................... 13
Associated documentation ......................................................... 13
2 Safety
2.1 Safety precautions .................................................................. 15
2.2 Safety labels ........................................................................... 16
2.3 Other warnings .......................................................................... 16
2.4 Emergency procedures .............................................................. 17
2.4.1
Emergency shutdown........................................................... 17
2.4.2
Power failure routine ............................................................ 17
2.4.3
Restart Procedure................................................................ 17
3 Preparing the gel
3.1 Introduction ............................................................................... 19
3.2 Bind-Silane treating glass plates ................................................. 19
3.3 Positioning of the reference markers .......................................... 20
3.3.1
Positioning before gel casting ............................................... 21
3.3.2
Positioning before gel scanning ............................................ 22
3.4 Gel casting ................................................................................ 22
3.5 Gel running conditions ............................................................... 23
3.6 Gel staining ............................................................................... 23
3.7 Gel scanning with the ImageScanner ......................................... 23
3.7.1
Gel position.......................................................................... 23
3.7.2
Scanning ............................................................................. 25
3.8 Gel scanning with the Typhoon ................................................. 26
3.8.1
SYPRO ruby stained gels...................................................... 26
3.8.2
SYPRO orange stained gels .................................................. 26
3.8.3
Image on screen after scanning ........................................... 26
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Contents
4 Selecting spots on the gel and
creating a pick list
4.1 Using ImageMaster 2D Elite ...................................................... 27
4.1.1
Simulating the picker head size ........................................... 27
4.1.2
Detection and editing of the markers.................................... 28
4.1.3
Detection and selection of spots for picking.......................... 30
4.1.4
Designating the reference markers....................................... 31
4.1.5
Creating the pick list ............................................................ 32
4.1.6
Saving the pick list file ......................................................... 33
4.2 Creating pick list files from alternative gel image
evaluation softwares .................................................................. 33
5 Setting up the Spot Picker
5.1 Select picker head ..................................................................... 35
5.2 Place the microplates ................................................................ 35
5.3 Place the gel ............................................................................. 36
5.4 Switch on mains power to the instrument ................................... 38
5.5 Start Ettan Spot Picker Control software ..................................... 38
5.6 Prime the system ...................................................................... 39
5.7 System set up ........................................................................... 40
5.7.1
Picker head versus gel Z position......................................... 41
5.8 Picking parameters ................................................................... 43
5.8.1
Recommended parameters ................................................. 43
5.8.2
Jazz .................................................................................... 43
5.8.3
Aspirate volume and Aspiration flow..................................... 44
5.8.4
Dispense volume and Dispense flow .................................... 45
5.8.5
Lift ...................................................................................... 46
5.8.6
Rinse volume and Rinse strokes .......................................... 46
5.9 Reference Marker area .............................................................. 47
5.10 Save and Exit System Setup ....................................................... 47
6 Picking a gel
6.1 Introduction .............................................................................. 49
6.2 Load a pick list .......................................................................... 50
6.3 Detection of the reference markers ............................................ 52
6.4 Pick the gel ............................................................................... 56
6.5 Pause picking ........................................................................... 56
6.6 Stop picking .............................................................................. 56
6.7 Changing microplates ................................................................ 57
6.8 After picking .............................................................................. 57
6.9 Repeating a picking run. ........................................................... 58
6.10 Resume picking after Ettan Spot Picker has been stopped ......... 59
6.11 Open result files ........................................................................ 61
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7 Picking a gel without a pick list
7.1 Introduction ............................................................................... 63
7.2 Manual picking directly from gel without a pick list ..................... 63
8 Troubleshooting
9 Staining protocols
9.1 Gel fixing ................................................................................... 69
9.2 SYPRO ruby .............................................................................. 69
9.3 SYPRO orange ........................................................................... 69
9.4 Silver staining compatible with MS analysis ................................ 70
9.4.1
Using the Modified PlusOne™ method................................. 70
9.4.2
Using the Hoefer automatic gel stainer ................................. 71
9.5 Coomassie staining .................................................................... 72
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List of figures
Fig 1-1.
Fig 3-1.
The Ettan Spot Picker system..................................................... 12
Position of the reference markers on the gel backing
when gel has been cast. ............................................................ 21
Fig 3-2.
Position of the reference markers on the gel backing
outside the gel. .......................................................................... 22
Fig 3-3.
Position of the reference markers below the gel backing............. 22
Fig 3-4.
Correct positioning of the gel on the ImageScanner. ................... 24
Fig 3-5.
Orientation of gel image before saving. ....................................... 26
Fig 4-1.
The use of Draw Spots pen tool to simulate the size
of the picker head. .................................................................... 28
Fig 4-2.
Correctly detected reference marker. ......................................... 29
Fig 4-3.
Incorrectly detected reference markers. ..................................... 29
Fig 4-4.
Correct appearance of the Measurements Window. .................... 33
Fig 4-5.
Correct table format and headings in Excel................................. 34
Fig 4-6.
Correct pick list format and headings. ........................................ 34
Fig 5-1.
Place the microplate racks......................................................... 35
Fig 5-2.
Place the microplates. ............................................................... 35
Fig 5-3.
Place gel tray............................................................................. 36
Fig 5-4.
Anchor the gel with the gel holders. ........................................... 37
Fig 5-5.
Correct position of gel and reference markers in the gel tray. ...... 37
Fig 5-6.
The System Set-up window........................................................ 40
Fig 5-7.
Setting the picker head height.
a: Picker head is too high,
b: Picker head is correct height,
c: Picker head is too low. ........................................................... 42
Fig 5-8.
Picking parameters.................................................................... 43
Fig 5-9.
Example of effect of jazz amplitude on the picking success rate.
A 1.4 x 1.2 picker head was used to pick from 1mm thick gels (12%T)
on glass backing........................................................................ 44
Fig 5-10. Example of effect of aspirate volume on the picking success rate.
A 1.4 x 1.2 picker head was used to pick from 1mm thick gels (12%T)
on glass backing........................................................................ 45
Fig 5-11. Example of effect of dispensing volume on the dispensing success
rate. A 1.4 x 1.2 picking head was used to pick from 1mm thick gels
(12%T) on glass and GelBond backing. ..................................... 46
Fig 6-1.
Main window before loading a pick list. ...................................... 50
Fig 6-2.
Loaded picklist. ......................................................................... 51
Fig 6-3.
Move camera............................................................................. 52
Fig 6-4.
Incorrect reference marker detection. ........................................ 55
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List of figures
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Introduction
1 Introduction
The Ettan™ Spot Picker is designed to automatically excise
polyacrylamide gel plugs, containing proteins from 2D gels, and then
transfer the plugs to microplates. These gel plugs are suitable for
further processing prior to MALDI-TOF mass spectrometry analysis.
The Spot Picker is compatible with 2D gels that have been post-stained
with Coomassie™, silver or SYPRO™ dyes, and for pre-labelled
proteins. Gels must be immobilized on plastic backing or glass.
Spots of interest can be evaluated and selected by using the
ImageMaster™ 2D Elite gel image analysis software. A pair of
reference markers must be included within the gel and also detected
during image analysis. These reference markers are used to enable the
Spot Picker to transform image (pixel) X-Y co-ordinates for each spot
into a millimetre position to pick from.
Following excision from the gel, the plugs are dispensed into 96-well
microplates, along with a volume of dispensing fluid. The Ettan Spot
Picker is capable of picking up to 9600 plugs in any single picking run.
1.1 The Ettan Spot Picker system
The Ettan Spot Picker system comprises the following:
• Ettan Spot Picker instrument
• Ettan Spot Picker Instrument Control Software, running under
Windows NT operating system on a PC
• PC with frame grabber card for video display
Fig. 1-1 shows the Ettan Spot Picker system.
A detailed description of the Ettan Spot Picker instrument is provided
in the Ettan Spot Picker Instrument Handbook.
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1
Introduction
Fig 1-1. The Ettan Spot Picker system.
1.2 Method overview
With the Ettan Spot Picker, gel plug picking can be defined in a number
of steps:
12
1
Preparing and pouring the gel onto a backing, including reference
markers. Alternatively, use precast gels and include the reference
markers before scanning.
2
Running the 2D separation.
3
Fixing and staining the gel after electrophoresis.
4
Scanning the gel including applied reference markers.
5
Analysis of the gel image, selecting the protein spots to pick and
creating the pick list file.
6
Loading the gel onto the Ettan Spot Picker and excising the selected
gel plugs into microplates.
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Introduction
1.3 The User Manual
The Ettan Spot Picker User Manual provides technical information
and operating instructions for the Ettan Spot Picker instrument and the
Ettan Spot Picker Instrument Control Software. Gel casting and gel
running are described briefly. In addition, instructions for scanners and
gel image evaluation softwares are included. For detailed information
on the instrument, the user is referred to the Ettan Spot Picker
Instrument Handbook.
1.4 Accessories
In addition to the items delivered with the system, Ettan Spot Picker
requires the following accessories:
• Gel scanner: ImageScanner™ or Typhoon™
• Gel image evaluation software: ImageMaster 2D Elite
• 96-well microplates
See the Ettan Spot Picker Instrument Handbook for ordering
information.
1.5 Associated documentation
The Ettan Spot Picker Instrument Handbook provides safety
instructions, technical information and basic operating instructions for
the Ettan Spot Picker instrument. In addition, maintenance schedules
and instructions for user maintenance are included.
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Introduction
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Safety instructions
2 Safety instructions
IMPORTANT! To avoid any risk of injury, the instrument should only
be operated by properly trained personnel and always in accordance
with the instructions provided.
The purpose of this chapter is to describe safety precautions and
present all safety labels that are attached to the instrument. Built-in
safety functions are described, and emergency and disposal procedures
are included.
2.1 Safety precautions
1
Read this entire manual before using the Ettan Spot Picker
instrument.
2
This instrument is designed for indoor use only.
3
The instrument must always be used with the protective earth lead
of the power cord correctly grounded to earth at the mains outlet.
4
To permit sufficient cooling, ensure that the vents at the top and
bottom of the instrument are not covered.
5
Do not operate the instrument in extreme humidity (above 95%).
Avoid condensation by letting the unit equilibrate to ambient
temperature.
6
Do not operate the instrument in direct sunlight.
7
Keep the instrument dry and clean. Wipe regularly with a soft damp
tissue. Let the instrument dry completely before use.
8
Any equipment connected to the instrument should meet the
requirements of the EN 61 010-1 or other international safety
standards.
WARNING! Do not use the Ettan Spot Picker in any other way than
described in this User Manual. The protection provided by the
instrument may be weakened if other operations are performed.
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2
Safety instructions
2.2 Safety labels
The following safety labels are attached to the Ettan Spot Picker
instrument to warn the user of potentially hazardous conditions:
At the picker head
Fig 2-1. Safety label located on the Y-arm, near the picker head.
WARNING! MOVING PARTS. The picker head and camera assembly
can make sudden, rapid movements. Keep all body parts clear when
the Spot Picker is in operation.
WARNING! Never put your hands underneath the picker head during
instrument operation. The picker head has the capacity to puncture
skin. In the event of an emergency, press the Stop button on the Spot
Picker: the instrument immediately ceases movement and all motors
are de-energized.
WARNING! To prevent fire or shock hazard, do not spill liquids into
the camera body.
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Safety instructions
2.3 Other warnings
WARNING! HEAVY OBJECT. The Ettan Spot Picker weighs about 40
kg. Two persons are required to lift the instrument.
WARNING! NO SERVICEABLE PARTS INSIDE. Do not open covers.
Service and maintenance should be performed by qualified
personnel.
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2
Safety instructions
2.4 Emergency procedures
2.4.1 Emergency shutdown
In the event of an emergency, press the Stop button on the Ettan Spot
Picker front panel. The instrument immediately ceases movement and
all motors are de-energized.
2.4.2 Power failure routine
In the event of a power failure, the experiment is interrupted in an
undefined state. However, the data for gel plugs picked up to the power
interruption is saved.
When power returns, the following will happen:
1
The picker head and the syringe pump (dilutor) are homed.
2
The PC starts and displays the log-in dialogue.
2.4.3 Restart Procedure
In the event of system shutdown due to power failure, emergency stop
or process interruption, malfunctions must be rectified before the Ettan
Spot Picker is restarted.
To restart the Ettan Spot Picker:
1
Check that the Power indicator on the Ettan Spot Picker instrument
lights.
2
Select Programs:Ettan Spot Picker:Ettan Spot Picker from the Windows
Start menu.
Reload the resultfile and continue picking, see instructions in Section
6.10. The last picked gel plug may be lost. This depends on the exact
moment when the interruption occurred.
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Preparing the gel
3 Preparing the gel
3.1 Introduction
Spot picking with the Ettan Spot Picker requires that gels are precast on
backing (e.g. Ettan DALT II Pre-cast Gel 12.5) or immobilized on
backing during casting. Two different types of backing may be used,
either: GelBond™ PAG film; or a glass plate, treated with a Bind-Silane
solution.
Note: To scan a gel with fluorescently labelled proteins, it is important
that GelBond is not used as the gel backing. The reason for this
is that GelBond is a plastic material and all plastics have intense
fluorescenses at the wavelengths used for scanning.
3.2 Bind-Silane treating glass plates
The following protocol for treatment of glass plates was optimized for
Bind-Silane from Amersham Pharmacia Biotech.
Note: It is important that glass plates are properly clean. Before re-use,
soak the plates overnight in a 5% DeconTM 90 solution. Do not
leave plates standing in a Decon solution for a longer time as this
will eventually cause etching due to the alkali nature of Decon.
1
Thoroughly wash the plate to be treated. Take care to remove any
gel fragments attached to the plate from previous gels. 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.
2
Thoroughly rinse the plates with ddH2O to remove Decon
3
Dry the plate using a lint-free tissue or leave them to air dry.
4
Prepare the Bind-Silane working solution, see Table 3-1 :
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Preparing the gel
Reagent
Quantity
Ethanol
8 ml
Glacial Acetic acid
200 µl
Bind-Silane
10 µl
Double distilled H2O
1.8 ml
Table 3-1. Bind-Silane working solution.
5
Pipette 2-4 ml (depending on plate size) 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 1-1.5 hours
6
Polish the plate with a lint-free tissue, moistened with a small
amount of double-distilled water or ethanol.
The gels will stay attached to the glass during electrophoresis, staining
procedures, scanning and storage.
3.3 Positioning of the reference markers
Spot picking with the Ettan Spot Picker requires the use of reference
markers. The reference markers are used to enable the Spot Picker to
transform image (pixel) X-Y co-ordinates for each spot into a
millimetre position to pick from. This enables picking from gels where
the protein spots are fluorescently labelled, as well as from gels where
the proteins have been stained with a visible stain.
The reference markers are designed so that they will be visible on the
scanned image of a gel (fluorescent or visible protein stain) as well as
being visible to the marker detection camera on the Ettan Spot Picker.
Two reference markers must be included in each gel that is to be picked
from.
The reference markers are self-adhesive and should be attached to the
gel backing (glass plate or GelBond film) prior to gel casting, or at the
latest, before the scanning of the gel. The reference markers can be used
in both densitometric and fluorescence scanning.
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Preparing the gel
3.3.1 Positioning before gel casting
It is important that the markers are appropriately placed on the gel
backing. The markers should be placed according to the following
protocol. Take care not to place the markers where they will interfere
with the pattern of protein spots in the gel.
1
Place the spacers on the edge of the treated glass plate/GelBond, to
ensure that the spacers do not cover the markers.
2
Place the marker approximately half-way along this edge, away
from the spacer, but not so far as to interfere with the protein spot
pattern.
3
Repeat steps 1 and 2 for the other edge of the gel backing.
4
When finished, the markers should be in positions similar to those
shown in Fig. 3-1.
Fig 3-1. Position of the reference markers on the gel backing
when gel has been cast.
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Preparing the gel
3.3.2 Positioning before gel scanning
If there is a sufficient backing outside the gel area, for example
Hoefer™ SE 600 gels, then it is possible to attach the reference markers,
after casting just before scanning.
Fig 3-2. Position of the reference markers on the gel backing outside the gel.
If there is not a sufficient backing-area outside the gel, it is possible to
attach the reference markers below the backing before gel scanning.
This is the recommended procedure for Ettan DALT II Pre-cast Gel
12.5.
Fig 3-3. Position of the reference markers below the gel backing.
Note: When attaching the reference markers on a gel backing after the
gel casting or electrophoresis run, the surface of the backing
must be completely dry, to ensure good adhesion of the markers.
3.4 Gel casting
Now assemble the gel holder for casting as described in the
manufacturers instructions. Pour the gels as soon as they are correctly
assembled.
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Preparing the gel
3.5 Gel running conditions
The gel being attached to a backing will not affect the standard gel
running conditions for a given sample. However, it is important that the
Immobiline™ DryStrip is correctly orientated. The Immobiline Dry
strip should be placed as normal (e.g. acidic end to the left), with the
gel backing lower-most.
3.6 Gel staining
To find examples of fixing and staining protocols, see Chapter 9
Staining protocols.
3.7 Gel scanning with the ImageScanner
3.7.1 Gel position
In order to secure the targeting accuracy of the Ettan Spot Picker it is
of particular importance to place the markers in the correct position
during scanning.
• The markers must be placed in the middle of the scanning area
(Fig. 3-4).
• The top of the gel, which was in contact with the Immobiline Dry
strip, must be placed on the right side when looking at the scanner
as showed in Fig. 3-4.
• The gels must be scanned upside-down, with the gel in contact with
the glass surface of the scanner, with a thin layer of water or gel
storage liquid between. The liquid layer will also make it easier to
lift the gel after scanning.
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Preparing the gel
Fig 3-4. Correct positioning of the gel on the ImageScanner.
24
1
Pour a few ml of gel storage liquid, or water, onto the glass surface
of the scanner.
2
Hold the gel (on backing) at an angle over the scanning surface.
3
To avoid air bubbles, slowly lower the gel until it is flat and resting
on the scanning surface.
4
To avoid seeing water drops on the gel image, wipe any drops off
from the upper surface of the gel backing with a lint-free tissue.
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Preparing the gel
3.7.2 Scanning
1 In the ImageMaster Labscan software, select the function Scan a
new image.
2
Select Refresh preview.
3
Select the scan area with the mouse arrow. Start in the lower left
position of the gel image on the screen. Drag an area to the upper
right position of the preview area, even if this area is not showing
the gel.
Note:
It is crucial to maximize the selected scan area in the upper
right position, before releasing the mouse button.
Otherwise, the calibration of the ImageScanner will be
affected, and resulting in imprecise spot picking.
4
Select the resolution: 300 dpi and Scan mode: transmissive.
5
Start scan.
6
After scanning, press the Rotation tool button, and rotate the image
counter clockwise, once. The resulting image will show the gel with
the acidic pH to the left, basic pH to the right and low Mw at the
lower end.
7
Save the image as a .tif file with File:Save as.
Note:
To secure the picking accuracy of the Spot Picker, no other
manipulations with the gel image is allowed (e.g. rotating,
or resizing).
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Preparing the gel
3.8
Gel scanning with the Typhoon
It is important to place the gel directly onto the scanner plate. Do not
reassemble.
The gel must be scanned upside-down, with the gel in contact with the
glass surface of the scanner, with a thin layer of water or gel storage
liquid between. The liquid layer will also make it easier to lift the gel
after scanning.
1
Pour a few ml of gel storage liquid, or water, onto the glass surface
of the scanner.
2
Hold the gel (on backing) at an angle over the scanning surface.
3
To avoid air bubbles, slowly lower the gel until it is flat and resting
on the scanning surface.
3.8.1 SYPRO ruby stained gels
It is recommended to use the green laser (532 nm), and the emission
filter of 610 nm with 30 nm bandpass. Use the platen focus setting.
3.8.2 SYPRO orange stained gels
The green laser (532 nm) is recommended. The emission filter of 580
nm with 30 nm bandpass should be selected. Use the platen focus
setting.
3.8.3 Image on screen after scanning
Before saving the image, the image on the screen has to be in the
orientation with the acidic pH to the left, basic pH to the right and low
Mw at the lower end.
Rotate the image to the specified position and save the image.
pH
Mw
Fig 3-5. Orientation of gel image before saving.
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Selecting spots on the gel and creating a pick list
4 Selecting spots on the gel and
creating a pick list
4.1 Using ImageMaster 2D Elite
The basis of spot picking with the Ettan Spot Picker is a pick list created
by an image analysis software. This section describes how to create this
pick list, using ImageMaster 2D Elite (V3.00 and higher).
Essentially, the pick list needs to contain the location, in pixels, of the
centre of each spot you wish to pick. It must also contain the pixel coordinates of the centres of the two reference markers.
This section assumes that the user is familiar with at least basic spot
detection and editing using ImageMaster. For more detailed
information on the use of ImageMaster, consult the User Manual.
4.1.1 Simulating the picker head size
When a spot boundary has been detected by ImageMaster or drawn by
the user, it is assigned a position in the image in terms of X and Y pixel
co-ordinates. This corresponds to the centroid of the spot (as defined
by the spot boundary). These are the co-ordinates used to define the
position that the picker head will remove a gel plug from. It is therefore
very important that spots are accurately detected so that the correct
area of gel is picked.
The following points should be considered during spot selection for
picking:
• The dimension of the picker head limits picking of spots very close
to each other. It is advisable to get an idea of the size of the picker
head in the gel image by using a pen tool of appropriate size (Fig.
4-1).
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Selecting spots on the gel and creating a pick list
• It may be preferable to manually specify the picking position for
spots with irregular or long drawn-out shapes and for very large
spots.
Fig 4-1. The use of Draw Spots pen tool to simulate the size of the picker head.
By using the Draw Spots tool, an approximate size of the picker head on
the scanned image can be seen. For a gel image which was scanned at
300 dpi, a pen size of 17 will represent a 1.4 mm diameter picker head.
Similarly, for a gel image scanned at 300 dpi, pen size 24 corresponds
to a 2.0 mm picker head.
4.1.2 Detection and editing of the markers
The two reference markers included in the gel must also be detected as
spots. Determining the centre of the marker is very important for
accurate picking. Often, the reference markers are not accurately
detected with the parameters used for detecting the protein spots in
your image. If the reference markers have not been correctly detected,
then follow these steps to define the reference markers.
Alternative 1
• Use the Grow Peaks tool and click inside the reference marker.
Alternative 2
28
1
Select a pen size slightly smaller than the size of the reference
marker.
2
Draw a spot on the reference marker, placing it centrally on the
marker.
3
Use the Grow Edge tool to fit the spot boundary to the boundary of
the marker.
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Note:
For accurate spot picking, it is essential that the reference
markers are correctly detected. When the edges of the
markers are not properly detected, the position on the pick
list given for the centre of the marker will be incorrect. This
will lead to an incorrect transformation of the spot coordinates from the pick list into mm positions for the picker
to pick from.
Make sure that the position-reference markers were detected as
distinct, circular spots and that the outline of the spot well corresponds
to the outline of the marker. This is illustrated in Fig. 4-2.
Fig 4-2. Correctly detected reference marker.
Drops of water below and above the gel during the scanning can affect
the detection as seen in Fig. 4-3, 2.
An imprecise use of the detection tools can also result in an incorrect
detection, see Fig. 4-3, 3
Fig 4-3. Incorrectly detected reference markers.
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Selecting spots on the gel and creating a pick list
4.1.3 Detection and selection of spots for picking
After performing spot detection and spot editing, ImageMaster
generates a measurements table containing spot related data.
When the measurements table is saved as a pick list, all the spots in the
table will be picked. Therefore, if only a selection of the spots are
requested to be picked, they have to be defined. This can either be done
manually or by using the spot filtering tools.
Manual selection of spots to be picked
Once the spot editing is satisfactory, go to the spot filtering mode.
1
Deselect all spots by pressing the Select/Deselect all button. All the
spots should appear blue. If they do not, press the button again.
2
Select the spots for picking by clicking on them with the left mouse
button.
3
The reference markers must also be selected.
4
In case of errors, click on the spot with the right mouse button to
deselect the spot.
5
Once this process is finished, see Section 4.1.5 Creating the pick
list.
Automated selection of spots to be picked
Once the spot editing and spot matching are satisfactory (they must be
complete to apply the automatic filtering), go to the spot filtering mode.
Decide to apply either comparison or range parameters. If parameters
are set in both comparison and range, they will both be applied when
the Apply Filter button is pressed, no matter which window is displayed.
• The Range parameters specify explicit ranges of values that a spot
must have in its measurement fields in order to be selected.
• The Comparisons parameters select spots based on comparisons
with spots that are matched to it in other gels.
For a detailed explanation of how to use these functions, please refer to
the ImageMaster reference manual and/or help function.
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After the automatic filter has been applied, the reference markers have
to be selected manually if they have not been automatically selected. At
this stage, it possible to manually edit the spot selections, as described
in the preceding section.
Once this process is finished, see Section 4.1.5 Creating the pick list.
4.1.4 Designating the reference markers
The reference markers must be distinguished from protein spots in the
pick list. This is achieved in ImageMaster by putting an entry into the
comment field of the reference markers.
1
In the main image window, select Edit:Edit Spot Fields.
2
Select the arrow tool, and click on the left hand side reference
marker.
3
In the Edit Spot Fields dialogue box, select the Field as comment.
4
Enter “IR1”(uppercase letters, with no space between) into the
Value field and press Set. This designates that the co-ordinates from
this spot represent the first reference marker.
5
With the selected arrow tool, click on the right hand side reference
marker.
6
Enter “IR2” into the Value field and press Set.
7
Press Done.
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8
To view these comments on screen, go to Tools:Options, select the
Display tab and tick the Data annotations box and set the field to
Comment.
9
Check that “IR1” and “IR2” are in the Comment column in the
Measurements window.
Note:
When using ImageScanner, it is strongly recommended that
reference markers are assigned in the manner described
above. This way, reference marker 1 will always be on the
left-hand side of the gel in the geltray (if the Immobiline
Dry strip is at the top) and marker 2 will always be on the
right. This will greatly aid identification of the different
markers.
4.1.5 Creating the pick list
The pick list is created from the measurements window. However,
before the pick list can be saved, the measurements table must be put in
the correct format, as described below. If the pick list is not saved in this
format, the picker will not recognise the file and will, therefore, not be
able to pick the spots.
To create the pick list in the correct format:
32
1
Select Tools:Options. When the options window appears, select the
Display tab. Set the Co-ordinate field to Pixels. Click OK.
2
Activate the Measurements Window.
3
Using the Select Fields button, make sure that only the Spot, Scaled
Coords (Pixels) and Comment fields are selected.
4
Deselect any other column by un-ticking the appropriate box.
5
If the order of the columns (left to right) does not appear the same
as in Fig. 4-4, use the Move Up and Move Down buttons in the field
selection box until they are correctly ordered.
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6
The position of the reference markers (spots 1 & 14 in Fig. 4-4) in
the pick list is unimportant.
Fig 4-4. Correct appearance of the Measurements Window.
4.1.6 Saving the pick list file
When the measurement window table is correctly formatted, the pick
list can be saved.
1
With the Measurements Window active, select Edit:Copy To File.
2
Enter a suitable file name for the pick list.
3
The pick list must be saved as a text (*.txt) file.
No further editing of the pick list should be done at this point.
If any errors are found in the pick list, it is preferable to go back to the
appropriate step and create a new pick list.
4.2 Creating pick list files from alternative gel image
evaluation softwares
Ettan Spot Picker Instrument Control Software can not handle original
pick list data from alternative 2D gel image evaluation softwares.
However, if the required data can be exported to Microsoft Excel™, it
will be possible to use the data for spot picking with Ettan Spot Picker.
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Selecting spots on the gel and creating a pick list
To create a pick list from an alternative 2D gel image evaluation
software:
1
Export the data to Microsoft Excel. The following data are
required:
• Spot identity
• The co-ordinates of the centre of the spots
• The co-ordinates must be in pixels
• The x and y co-ordinates must have separate columns
2
Edit the data in Excel to create a table in a correct format:
• The table must contain only four columns
• The headings of the columns must be as follow:
id, x, y, comment
• The comment column must contain the IR1 and IR2 annotations
for the reference markers. If the 2D gel image evaluation
software does not allow exporting designations, the IR1 and IR2
annotations must be assigned manually to the appropriate spots.
Fig 4-5. Correct table format and headings in Excel.
3
Save the file (File:Save as) as a Tab delimited text (.txt) file.
Fig 4-6. Correct pick list format and headings.
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5 Setting up the Spot Picker
5.1 Select picker head
If the picker head needs to be changed, see instructions in the Ettan
Spot Picker Instrument Handbook.
5.2 Place the microplates
1
Place the microplate racks in the Spot Picker.
Fig 5-1. Place the microplate racks.
2
Place 1-4 microplates in the plate tray with A1 position in the front.
Fig 5-2. Place the microplates.
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Setting up the Spot Picker
5.3 Place the gel
1
Place the gel tray in the Ettan Spot Picker. The guide feet on the tray
should fit onto pins on the locator plate; it is only possible to fit the
gel tray in one direction.
Fig 5-3. Place gel tray.
2
36
Place the gel in the gel tray and cover with a suitable fluid (e.g.
fixing/preservative liquid or double-distilled water) to a surface
above the gel of approximately 2 mm. If many spots will be picked,
a larger volume of fluid may be required to compensate for
evaporation.
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Note:
3
To avoid air bubbles, place fluid in the tray before the gel.
The gel is then placed in the tray angled to the buffer
surface.
Position the gel so that the reference markers lie within the parallel
lines in the centre of the gel tray (see Fig. 5-5). The reference
markers must not cross or touch the lines because this will affect the
reference marker detection.
Fig 5-4. Anchor the gel with the gel holders.
4
Assemble the gel holders within the slots in the tray. Anchor the gel
down firmly with the gel holders, tightening the screws no more
than finger tight.
Note:
For large gels, the gelholders should be positioned in the
geltray before the gel is placed. Otherwise, the gel could be
damaged by the gel holders.
Fig 5-5. Correct position of gel and reference markers in the gel tray.
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Setting up the Spot Picker
5.4 Switch on mains power to the instrument
Turn on mains power to the instrument with the mains switch located
on the back of the instrument.
5.5 Start Ettan Spot Picker Control software
1
To start the Ettan Spot Picker Instrument Control Software, select
Programs:Ettan Spot Picker:Ettan Spot Picker from the Windows Start
menu:
2
A dialogue regarding Homing of instrument will be shown.
Press OK.
WARNING! MOVING PARTS. The picker head and camera assembly
can make sudden, rapid movements. Keep all body parts clear when
the Spot Picker is in operation.
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5.6 Prime the system
Place the buffer tubing in a suitable buffer for spotpicking. When the
picklist is loaded, the software will inform the user of the volume of
buffer required to pick all spots. It is recommended that either doubledistilled water or the appropriate wash/destain solution for the gel
plugs is used as the system buffer.
Priming the syringe removes air bubbles in the spot picker tubing and
also rinses the tubing with fresh solution. Priming should be made
when tubing, bottles or buffer have been changed.
Note: It is important to ensure that the position of the rinse station is
correctly set in the System Setup (see Ettan Spot Picker
Instrument Handbook) before priming the system.
1
Select Tools:Prime Syringe.
2
In the Prime Syringe window, enter the number of priming strokes
(5-10 priming strokes are recommended). Press Prime.
3
The Stop button can be used to interrupt the priming.
4
When the priming is completed, press Exit.
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Setting up the Spot Picker
5.7 System set up
Select System:System Setup.
Fig 5-6. The System Set-up window.
The System Setup screen allows the user to set:
• The position of the picker head according to microplates and rinse
station. For instructions, see the Ettan Spot Picker Instrument
Handbook.
• The height of the picker head versus the gel backing material.
• The parameters used for picking the gel plugs.
• The reference markers area ranges, which are used by the detection
software.
At any stage, the user can decide to abandon the System Setup
procedure by pressing the Cancel button. If the Cancel button is pressed,
any changes made to the System Setup will be lost. To retain new System
Setup parameters, press the Save & Exit button.
It is advisable to press the Initialize Instrument and Go to Home buttons
prior to setting any of the system locations.
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The blue arrows visible on the right hand side of the screen are used to
manoeuvre the picker head to various positions. Once the picker head
is placed appropriately (e.g. in the rinse station), the location can be
stored by pressing the appropriate Set button.
The blue arrows are used to move the picker head in the X/Y/Z
directions. By pressing the buttons on the arrows, the head will move
stepwise in the corresponding directions. As long as a button is pressed,
the head will keep moving.
For the Move X/Y arrow, the outermost buttons will move the head in 10
mm steps, the middle buttons in 1 mm steps and the innermost buttons
in 0.1 mm steps.
The Move Z arrow buttons will move the head in 5, 1 and 0.1 mm steps,
respectively.
CAUTION! Be careful when moving the picker head, because if the
head hits an obstacle, the motor will shut-off and the instrument will
be re-initialized. Similarly, rapidly moving the picker head into an
object has the potential to damage the picker head.
5.7.1 Picker head versus gel Z position
This value indicates the Z-height at which the picker head meets the gel
backing. This must be correctly set for maximum picking efficiency.
Note: This value must be checked before every picking run.
To set the correct Z-height of the picker head:
1
Use the Move X/Y blue arrows to move the picker head to an area
where there is gel backing, but no gel, or no important area of the
gel.
2
Use the Move Z arrows to carefully lower the picker head. Using the
innermost button will move the picker head in 0.1mm steps.
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Setting up the Spot Picker
3
When the tip of the picker head touches the backing, you will see a
gap between the holder and the picker head piston.
a
b
1-2 mm
c
>2 mm
Fig 5-7. Setting the picker head height.
a: Picker head is too high,
b: Picker head is correct height,
c: Picker head is too low.
4
If a gap does not form (Fig. 5-7a), the tip of the picker head is not
touching the gel backing. This will result in the gel plugs being left
in the gel or picked partially.
If a gap greater than 2 mm can be seen (Fig. 5-7c), the picker head
is pressing down too hard on the gel backing. This will result in a
shorter life span for the picker head and has the potential to scratch
glass plates.
The correct position for the picker head is with a 1-2 mm gap
showing at the top of the piston (Fig. 5-7b).
5
42
Press the Set Gel-Z Coordinate when the Z position of the picker head
is correct.
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5.8 Picking parameters
There are various parameters that must be set correctly for successful
picking and dispensing of gel plugs. It is difficult to give exact values for
some of them because the optimal values may differ depending on types
of backing, percentage gel and staining methods. The parameters can
be varied in experiments to optimize the picking from different gel
types.
Fig 5-8. Picking parameters
5.8.1 Recommended parameters
Out of performed experiments, some guidelines and recommendations
for different type of gels and picker heads have been compiled.
Gel
Picker
head
Recommended parameters
Thickness
Backing
Type
Jazz
Asp.vol.
Asp. flow
Disp.vol.
Disp.flow
Lift
1 mm
glass
1.4 x 1.2
2.0 x 1.2
0.9-1.1
1.0-1.2
15-50
30-50
20
20
100-250
150-250
30
30
0
0
GelBond
1.4 x 1.2
2.0 x 1.2
0.7-1.0
1.1-1.3
15-50
30-50
20
20
100-250
150-250
30
30
0
0
glass
1.4 x 1.7
2.0 x 1.7
1.2-1.4
1.2-1.4
25-50
25-50
20
20
100-250
150-250
30
30
0
0
1.5 mm
Table 5-1. Recommended picking parameters.
5.8.2 Jazz
The Jazz parameter determines the amount of sideways movement that
the picker head will perform at its lowest Z-position to shear the gel
plug from the gel backing. A Jazz of 1 mm corresponds to a movement
of 1 mm both in the plus and minus direction along the X-axis of the
spot picker.
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The amplitude of the Jazz depends primarily on the picker head size and
is also dependent on:
• Type of backing
• Quality of the Bind-Silane treatment
• Percentage of polyacrylamide used
• Gel thickness
Fig 5-9. Example of effect of jazz amplitude on the picking success rate.
A 1.4 x 1.2 picker head was used to pick from 1mm thick gels (12%T) on glass
backing.
5.8.3 Aspirate volume and Aspiration flow
The function of aspirating is to suck up the gel plug and keep it in place
inside the picker head tip, prior to dispensing into the microplate.
The Aspirate Vol. should be large enough to create an adequate negative
pressure in the inside of the picker head. Values between 15 and 50 µl
have been found to work very effectively. If this value is set too low,
then the gel plugs will be left behind in the gel. Values that are too high
may lead to some damage of the gel plugs.
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Fig 5-10. Example of effect of aspirate volume on the picking success rate.
A 1.4 x 1.2 picker head was used to pick from 1mm thick gels (12%T) on glass
backing.
The Aspiration Flow determines the rate of aspiration. A value of 20 ml/
min has been shown to work effectively. If this value is set too low, then
the gel plugs will be left behind in the gel. Values that are too high may
lead to some damage of the gel plugs.
5.8.4 Dispense volume and Dispense flow
Each gel plug is ejected from the picker head along with a volume of
fluid. The fluid serves two purposes. Firstly, it physically forces the gel
plug out of the picker head. Secondly, if a suitable buffer is used, this
can form part of the gel plug washing/destaining procedure.
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The Dispense Vol. should be large enough to ensure that each gel plug is
successfully ejected from the picker head. If this value is set too low,
then gel plugs will not be dispensed into the microplate and will be lost
when the picker head is next rinsed. The dispense volume should not be
below 100 µl.
Fig 5-11. Example of effect of dispensing volume on the dispensing success rate.
A 1.4 x 1.2 picking head was used to pick from 1mm thick gels (12%T) on glass
and GelBond backing.
The Dispense Flow needs to be high enough to rapidly eject the plug from
the picker head. It is recommended that the dispense flow value is set
to max. 30 ml/min.
5.8.5 Lift
The Lift option allows you to raise the picker head from the gel backing
prior to aspirating the gel plug into the head. Under normal conditions
no lift is required. In some cases introducing a lift may actually reduce
the picking efficiency.
5.8.6 Rinse volume and Rinse strokes
The Rinse Volume and Rinse Strokes parameters determine the efficiency
of picker head washing, after each gel plug is picked. Under normal
conditions, a single syringe stroke with a volume 100-950 µl is
sufficient. Using more rinse strokes will increase the length of the
picking runs.
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5.9 Reference Marker area
The reference markers area is set in the Marker Area section of the System
Setup window. The two values (minimum and maximum) stipulate the
size (in pixels) of the reference markers. These two values are used
during detection of the reference markers to evaluate the objects seen
by the camera.
The default values for these parameters are Min Area: 30 000 and Max
Area: 46 000.
5.10 Save and Exit System Setup
Click on Save & Exit when the system configuration is completed. The
same settings will be used until altered by the user.
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6 Picking a gel
6.1 Introduction
For a successful picking run, it is assumed that all the necessary
preceding steps have been carried out in accordance with the
instructions in this User Manual and Ettan Spot Picker Instrument
Handbook.
A gel is now placed into the picker and a pick list has been created.
This section describes the actions to perform before, during and after
picking:
• Loading of a pick list
• Detection of the reference markers
• Start picking
• Stop/pause picking
• Changing microplates
• Procedure after picking
• Handling of result files
The section also describes the resuming of picking runs:
• When unpicked gel plugs have been located
• When a run has been stopped
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Fig 6-1. Main window before loading a pick list.
Before proceeding, it is advisable to press the Initialize Instrument and Go
to Home buttons.
6.2 Load a pick list
1
50
In the Load Pick List window, select the type of used reference
markers (Black/White). Press the Load Pick List button.
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2
Browse for the pick list.
3
Select the pick list.
4
If the gel has been scanned with an ImageScanner, check the Use
Correction box. Select the used scanner in the Scanner Id list box, and
select the used resolution when the gel was scanned in the Resolution
(DPI) list box.
5
Click on Open.
6
The pick list data will appear on screen and a representation of the
spots to be picked will be drawn in the main part of the window. X
and Y coordinate columns are empty, because the reference
markers have not been detected yet.
Fig 6-2. Loaded picklist.
7
In the Load Pick List window, press Next.
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8
If microplates have not been previously loaded, label the
appropriate number of microplates (as shown on screen) and load
the first four (or fewer) onto the Spot Picker microplate rack (See
Section 5.2). If a gel has not been loaded, place the gel according to
Section 5.3.
9
Press Next.
6.3 Detection of the reference markers
1
In the Find First Marker (IR1) window, press Move to First Marker.
2
Move the camera with the blue arrows to a position over the first
reference marker (IR1) on the gel.
Fig 6-3. Move camera.
52
3
If the reference marker is underneath the camera, but not visible in
the Camera view window, use the Contrast and Threshold sliding bars
to find an image of good quality. If the image appears blurry, adjust
the camera aperture and focus until the marker image is sharp in
the Camera view.
4
If the marker is visible in the Camera view window, but there is no
image in the Marker detection window, first adjust the positions of
the Contrast and Threshold sliding bars until an image is visible in the
Marker detection window. If adjusting the sliding bars does not lead
to an image becoming visible in the Marker detection window, set the
Contrast and Threshold sliding bars to their central positions. Then
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adjust the camera aperture until the marker is visible in the Marker
detection window.
5
When the majority (or all) of the reference marker is displayed in
the Marker detection window, the perimeter of the window will
change from red to green. This indicates that there is a stable
detection of the reference marker. Go to step 9.
6
If the majority or all of the reference marker is visible in the Camera
view window, and the perimeter of the Marker detection window
remains red, then make sure that the Select Marker Type box in the
Load Pick List window has the correct colour of marker selected. Use
the Back button to go to the Load Pick List window. Use the Next
button to return to the Find First Marker (IR1) window. When the
perimeter is green, go to step 9.
7
If there is a portion (or none) of the reference marker visible in the
Marker detection window and the perimeter of the Marker detection
window is red, this means that the camera is not sufficiently centred
over reference marker.
• Adjust the position of the camera using the blue camera
movement arrow.
• Return to step 3 and follow the instructions from there onwards
until there is a enough of the reference marker visible in the
Marker detection window to cause the perimeter to turn green.
8
When the perimeter of the Marker detection window is green, the
Spot Picker has detected a stable image of the reference marker.
9
Below the Marker detection window, there are two boxes which
show the offset of the centre of the marker from the centre of the
camera.
10 Press the Adjust button and the reference marker will be centered in
the Marker detection window. The marker is assumed to be centred
when the values in both offset boxes are in the range -0.1 to +0.1.
The procedure may need to be repeated until the offset values fall
within the acceptable range.
Note:
Achieving 0.0 offset values is not required. The software
accounts the offset values so that the picking precision will
not be affected.
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11 When the offset values are in the range –0.1 to +0.1 press the Move
to First Marker button.
Note:
It is only when the Move to First Marker button is pressed that
the picker records the position of the marker.
12 Press Next.
13 In the Find Second Marker (IR2) window, press Move to Second Marker
14 Move the camera with the blue arrows to a position over the second
reference marker (IR2) on the gel.
15 When the perimeter of the Marker detection window is green, the
picker has detected a stable image of the reference marker.
16 If the reference marker is not visible in any of the windows or the
perimeter is red, see the step 3-7.
17 Press the Adjust button and the reference marker will be centered in
the Marker detection window. The marker is assumed to be centred
when the values in both offset boxes are in the range -0.1 to +0.1.
The procedure may need to be repeated until the offset values fall
within the acceptable range.
18 When the offset values are in the range –0.1 to +0.1, press the Move
to Second marker button.
19 In the Find Second Marker (IR2) window, press Next.
The software will now calculate the coordinates for the spots to be
picked, and the pick list table on the screen will be updated.
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Note: Damaged reference markers and impurities in the gel image can
result in incorrect detections. Also, reflections, shadows and
incorrect set contrasts, thresholds and camera aperture can result
in similar errors. This will result in imprecise picking.
Fig 6-4. Incorrect reference marker detection.
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6.4 Pick the gel
1
In the Pick Spots window, press Pick.
2
In the Result File Location window, select the location for the picking
results file(s).
3
Enter output directory name and user name. The results will be
saved as a folder in the selected location. Each plate filled (even if
only partially) during the picking run is given a unique results file.
4
Click on Create Directory & Start Picking.
6.5 Pause picking
Press the Pause button, if the picking procedure has to be paused to fill
the buffer reservoir, for example. When the Pause button is pressed, the
picker will pause at the end of a specific operation.
Press the Continue button to resume picker movement.
6.6 Stop picking
Press the Stop button of the instrument, or the Stop button in the main
application window, if the Spot Picker needs to be immediately
stopped.
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Picking a gel
There is a facility to restart a picking run after pressing Stop. This
procedure is described in section 6.10.
6.7 Changing microplates
If a picking run consists of more than 384 gel plugs, the software will
instruct the user to change plates.
1
Remove the four full plates and cover them with plastic lid or
adhesive foil cover.
2
Place the required number of labelled microplates onto the Spot
Picker.
3
Press New Plate.
This event will happen every time four full microplates are left on the
spot picker.
6.8 After picking
When the picking run is completed:
1
Remove the gel from the gel tray and store/discard as appropriate.
2
If the gel is to be discarded, scrape the gel off the glass plate with a
plastic spacer or similar object. Then put the plates into a 5%
Decon solution overnight to remove any gel fragments which
adhere to the plate.
3
Rinse the buffer lines with ddH20 using the prime syringe tool.
4
Rinse the liquid out of the gel tray with ddH20 and leave the gel
tray to dry.
5
Exit the Ettan Spot Picker Instrument Control Software and turn
the computer and the Spot Picker off.
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6
Picking a gel
6.9 Repeating a picking run.
In case of unsuccessfully picked gel plugs, it is possible to repeat a run
to complete the picking. The picking parameters may need to be
changed, e.g. increase the jazz.
1
Note the positions of microplates and wells with missing gel plugs.
2
In the Load Pick List window, press Load Pick List.
3
Browse for the result of the performed picking run (extension .mfl).
4
Select the result file. Do not use the correction function again!
Click on Open.
5
The result table will be shown on the screen.
Note:
6
58
The detection of reference markers has to be done again.
The Picker X and Y values are missing in the table.
Perform detection of reference marker, see Section 6.3
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6
Picking a gel
7
In the updated result table, select the rows for each unpicked gel
plug and the ticks in the table will be replaced by filled circles.
8
Press Pick. The user will be informed by the software to place the
specific plate in position 1.
Note:
9
It is important to remove the volumes of liquid in the empty
wells otherwise the volume will be too high after repicking.
Press OK and the picking run will start.
6.10 Resume picking after Ettan Spot Picker has been
stopped
1
When the Ettan Spot Picker has been stopped by user or due to a
technical error, an information message appears. Press OK.
2
In the Load Pick List window, press Load Pick List.
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6
Picking a gel
3
Browse for the result of the interrupted picking run
(extension .mfl).
4
Select the result file. Do not use the correction function again!
Click on Open.
5
The result table will be shown on the screen. Ticked positions mean
picked gel plugs. Filled circles represent unpicked gel plugs.
Note:
60
The detection of reference markers has to be done again.
The Picker X and Y values are missing in the table.
6
Perform detection of reference marker, see Section 6.3.
7
Press Pick. The user will be informed by the software to place the
microplates. The partially dispensed micro plate and the next
undispensed microplates must be placed in their original positions.
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Picking a gel
Note:
Depending on exactly when picking was interrupted, the
last gel plug being picked may be lost.
6.11 Open result files
The format for the file names is Directory name_XX.apb, where the
directory name is the same as the name entered in the Output Directory
Name field and the XX is the number of the plate produced in that
picking run.
1
To view result files associated with each microplate of gel plugs,
select Tools/Output File Viewer.
2
To open a plate file, press the Open File button in the lower end of
the dialogue.
3
Browse and select the file to be viewed and click Open.
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6
Picking a gel
62
4
The viewer allows the user to look at the processing status of the
gel plugs in that micro plate.
5
To export data, press the Export Data button.
6
Save the data as a text file which may then be used in other
applications such as Microsoft Word™ and Microsoft Excel.
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7
Picking a gel without a pick list
7 Picking a gel without a pick list
7.1 Introduction
The Ettan Spot Picker allows spot picking in a mode similar to
manually picking the spots. This feature is useful, when there are a
limited number of spots to pick. The gel does not need to be scanned,
and no pick list must be prepared.
The picking will not produce a result file, so the user must keep track
of the spots.
Note: Use the delivered white sheet as a background to obtain a better
contrast of the gel image in the camera view.
7.2 Manual picking directly from gel without a pick list
The Ettan Spot Picker, gel and microplates must have been prepared
according to Chapter 5 Setting up the Spot Picker.
The function uses the picking parameters as set in the System setup.
Therefore, these need to be set correctly before using Click n’Pick.
1
Press Initialise Instrument and Go to Home.
2
Select Tools:Click n’Pick.
3
Select the Picker Head Size. This will alter the size of the circle in the
camera view window.
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7
Picking a gel without a pick list
4
The first gel plug will be dispensed in microplate well A1. However,
the position can be edited in the Next Well window
5
Set Contrast, Brightness and camera aperture, to achieve a good
image quality.
6
Move the square in the upper right window with the mouse pointer
to a preferred position of the gel.
7
Move the circle in the camera view by using the mouse pointer.
When a requested gel spot is detected within the picker circle, press
Pick.
Tip! It may be convenient to place e.g. a pen close to the spot which
will help to find it in the camera view. Do not activate the
movements of the picker during this procedure!
Note:
When the picker head has dispensed the gel plug into the
micro plate, the camera will move back to the picked
position.
WARNING! MOVING PARTS. The picker head and camera assembly
can make sudden, rapid movements. Keep all body parts clear when
the Spot Picker is in operation.
8
If several gel plugs from a spot are wanted to be pooled in the same
microplate well, it is possible by checking the Dispense in Single Well
box in the Next Well window. This has to be done before the first
picking to that well.
9
When the microplate is full. Replace the plate with an empty plate,
and press New Plate.
10 When the picking run is completed. Press Exit.
64
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8
Troubleshooting
8 Troubleshooting
To avoid errors and to obtain maximum picking success rate, it is
important to follow all the instructions in the User Manual and
Instrument Handbook.
The user must ensure that:
• The picker head is not damaged, and is clean on both the inner and
outer sides.
• The O-ring is present, and not damaged.
• Tubing is clean, and not damaged.
• The Z-height values are correctly set.
• The System setup parameters are correctly set.
Problem
Probable cause
Remedy
Gel plugs not removed from
gel.
The set jazz amplitude is too
low.
Increase the jazz value
stepwise with 0.1 mm
increments.
Inefficient aspiration.
The aspiration volume should
be minimum 15 µl. Increase
the aspiration volume. Check
the O-ring of the picker head
for damage. Check all tubing
for leaks and air bubbles.
Bind-Silane treatment was too
effective.
Reduce the concentration of
Bind-Silane.
The picker head does not reach
the gel.
Check the picker head’s Zheight versus gel.
The picker head is worn-out.
Replace the picker head.
A position, close to the current
gel plug's, has previously been
picked.
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8
Troubleshooting
Problem
Probable cause
Remedy
Gel plugs are picked but
deformed or not complete.
Only the upper part is picked
due to the picker head not
reaching the gel backing.
Adjust the picker head versus
gel Z-position.
The inner side of the picker
head is dirty.
Clean the picker head (both
inner and outer sides).
Gel plug is situated in a nearby
well. The microplate Z-height
has been too high so the
dispense flow has splashed it
into a nearby well.
Decrease the microplate Zheight.
Gel plug is in rinse station due
to adsorption to the outer side
of the picker head during
dispensing. The dispensing
height has been set too low.
Increase the microplate Zheight. Clean the outer side of
the picker head to reduce
liquid adsorption.
Gel plug is in rinse station due
to adsorption to the outer side
of the picker head during
dispensing. The dispensing
volume has been set too low.
Increase the dispensing
volume. Clean the outer side of
the picker head to reduce
liquid adsorption.
Gel plug is lost during transfer
from gel to microplate.
Increase the aspiration volume.
The identities of the two
reference markers have been
mixed up.
Confirm that the first detected
marker is the correct one on
the gel image.
Gel from spot has been picked
but is not found in microplate
well.
Very imprecise picking results.
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Troubleshooting
Problem
Probable cause
Remedy
Picking position errors.
Camera is not correctly
calibrated.
Check that the calibration has
been performed according to
the Instrument Handbook.
Incorrect detection of the
reference markers.
Be sure to have a distinct
circular picture of the marker
both in the camera and marker
detection view.
Uncorrected ImageScanner
image.
Scan according to User Manual
and use the correction option
when the pick list is imported.
There is no calibration file for
the used scanner.
Contact APBiotech Service for
calibration of the scanner.
The gel image has too low
resolution.
Scan the gel with higher
resolution, for example 300
dpi.
Picking errors for oval and
irregular shaped spots
The centre of the detected spot
in the 2D-software differs from
the preferred picking position.
Detect these types of spots
manually. For example with the
Grow Peaks tool or the Pen
tool.
Scratches on the gel glass
plates after picking.
The Z-height of the picker
head versus gel is too low.
Adjust the Z-height according
to User Manual.
The gel loosens from the glass
plate.
Inefficient Bind-Silane
treatment.
Prepare according to
recommendations in User
Manual. Clean the plates
properly and leave them in 5%
Decon over night.
The gel loosens from the glass
plate in a specific area during
picking.
Too many gel plugs have been
picked from a smaller area.
The gel cracks and become
damaged close to the picked
gel plug.
Gel stored in ethanol and
acetic acid has a tendency to
crack. This will not affect the
picking result, if not too many
spots are picked from the same
area.
Ettan Spot Picker User Manual 18-1147-61 Edition AA
Storage of the gel in double
distilled water reduces the risk
of cracking.
67
8
Troubleshooting
68
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9
Staining protocols
9 Staining protocols
9.1 Gel fixing
When the gel run is complete, the gel must be placed into a suitable
fixing solution as soon as possible. This will minimize protein spot
diffusion. As the gel is attached to a backing, a fix solution that
dehydrates the gel too quickly will cause the gel to crack and peel away
from the backing.
It is recommended that a solution of 30% ethanol and 7.5% acetic
(ethanoic) acid is used to fix gels. The gels must be left in this fix
solution for at least two hours. Leaving gels in this fix for longer
periods of time appears to have no detrimental effects on the gel.
9.2 SYPRO ruby
1
Fix the gel in 10% methanol and 7% acetic acid for at least 2
hours.
2
Place the gel directly into a polypropylene, polycarbonate or
polyvinyl chloride tray.
3
Cover the gel with the SYPRO ruby stain.
4
Incubate the gel for five hours - overnight with gentle shaking,
protected from the light.
5
Pour off the SYPRO ruby.
6
Wash the gel in 10% methanol, 6% acetic acid for 1-2 hours.
9.3 SYPRO orange
1
Place the gel directly into a polypropylene, polycarbonate or
polyvinyl chloride tray.
2
Prepare 250 ml of a 1:5000 dilution of SYPRO orange in 7.5%
acetic acid.
3
Stain the gel for three hours - overnight with gentle shaking,
protected from the light.
4
Wash the gel in 7.5% acetic acid for 60-90 minutes.
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9
Staining protocols
9.4 Silver staining compatible with MS analysis
9.4.1 Using the Modified PlusOne™ method
Allow at least 250 ml of each solution per gel.
All steps are carried out at room temperature with gentle shaking.
Make all solutions freshly when needed. Make sure to use only double
distilled (18.2 MΩ) water. All solutions should appear clear and
colourless before they are poured onto the gel.
Reagent
Quantity
Ethanol
75 ml
5% Sodium thiosulphate
solution
10 ml
Sodium acetate
17 g
ddH20
To a final volume of 250 ml
Table 9-1. Sensitizing solution.
Reagent
Quantity
Silver nitrate
625 mg
ddH20
To a final volume of 250 ml
Table 9-2. Silver nitrate solution (0.25%).
Reagent
Quantity
Sodium carbonate
6.25 g
Formaldehyde
100 µl
ddH20
To a final volume of 250 ml
Table 9-3. Developing solution.
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Staining protocols
Reagent
Quantity
EDTA
3.65 g
ddH20
To a final volume of 250 ml
Table 9-4. Stop solution.
1
Sensitize the gel in Sensitizing solution for 30 min.
2
Rinse the gel three times in ddH2O, for five minutes each rinse.
3
Incubate in a 0.25% silver nitrate solution for 20 minutes.
4
Rinse the gel twice in ddH2O, for five minutes each rinse.
5
Incubate the gel in Developing solution for up to 10 minutes. If the
staining is intense enough before the end of 10 minutes, move
directly to the stop solution.
6
Pour the developing solution off and add the Stop solution,
incubate for 10 minutes.
7
Rinse the gel three times in ddH2O, for five minutes each rinse.
8
Store the gel in ddH2O.
9.4.2 Using the Hoefer automatic gel stainer
The following method was developed for 1 mm thick 12% acrylamide
gels. Other thickness and percentage acrylamide gels may require
further optimization. The wash steps which follow the sensitizing and
silver stages are crucial for a low background to the gel.
Make all solutions freshly when needed. Make sure to use only double
distilled (18.2 MΩ) water. All solutions should appear clear and
colourless before they are poured onto the gel.
Reagent
Quantity
Sodium thiosulphate
500 mg
ddH20
To a final volume of 250 ml
Table 9-5. Sodium thiosulphate solution (0.2%).
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9
Staining protocols
Reagent
Quantity
Glacial acetic (ethanoic) acid
12.5 ml
ddH20
To a final volume of 250 ml
Table 9-6. Stop solution.
1
Fix the gel for 2 hours minimum in 30% ethanol, 7.5% acetic acid.
2
Wash the gel four times in 50% methanol, for eight minutes each
rinse.
3
Sensitize for 30 minutes in 0.2% sodium thiosulphate.
4
Wash the gel five times in ddH2O, for 20 minutes each rinse.
5
Incubate in 0.25% silver nitrate solution (Table 9-2 ) for 30
minutes.
6
Wash the gel twice in ddH2O, for four minutes each rinse.
7
Incubate in developing solution (Table 9-3 ) for 2-5 minutes.
8
Incubate in Stop solution (Table 9-6 ) for 60 minutes.
9
Store the gel in ddH2O.
9.5 Coomassie staining
72
1
Fix the gel for 60 minutes in 30% ethanol and 7.5% acetic acid.
2
Stain the gel for 60 minutes minimum in 0.1% Coomassie R-250 in
40% ethanol and 10% acetic acid. Use a Stock solution of 1 w/
vol.% Coomassie R-250 in 96% ethanol.
3
Destain the gel in 20% ethanol and 5% acetic acid.
4
Store the gel in ddH2O.
Ettan Spot Picker User Manual 18-1147-61 Edition AA
Index
IX
Index
A
aspirate volume ....................................................................... 44
aspiration flow ......................................................................... 45
B
Bind-Silane treatment .............................................................. 19
C
camera offset .......................................................................... 53
camera view window ................................................................ 52
contrast ................................................................................... 52
Coomassie staining .................................................................. 72
D
dispense flow .......................................................................... 46
dispense volume ..................................................................... 46
E
Ettan DALT II Pre-cast Gel 12.5 ............................................... 19
Ettan Spot Picker compatibility ................................................. 11
Ettan Spot Picker Instrument Control Software ......................... 11
start ................................................................................. 38
Ettan Spot Picker Instrument Handbook .................................. 13
Ettan Spot Picker system ......................................................... 11
G
gel image evaluation software .................................................. 13
gel running conditions ............................................................. 23
gel scanner ............................................................................. 13
ImageScanner .................................................................. 23
Typhoon ........................................................................... 26
gel tray .................................................................................... 36
GelBond PAG film ................................................................... 19
I
ImageMaster ............................................................ 11, 13, 27
ImageScanner ..................................................................13, 23
J
jazz ......................................................................................... 43
L
lift ........................................................................................... 46
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IX
Index
M
mains power ........................................................................... 38
MALDI-TOF ............................................................................. 11
manual picking ....................................................................... 63
marker detection window ........................................................ 52
microplate ............................................................................... 35
microplate rack ....................................................................... 35
P
pick list
creating ........................................................................... 32
from alternative gel image evaluation softwares ................. 33
loading ............................................................................ 50
regenerating .................................................................... 58
saving .............................................................................. 33
picker head
selecting .......................................................................... 35
simulating size ................................................................. 27
size circle ........................................................................ 63
z-position vs gel ............................................................... 41
picking
pause .............................................................................. 56
resume ............................................................................ 59
start ................................................................................. 56
stop ................................................................................. 56
picking parameters ................................................................. 43
precast gels ............................................................................ 12
prime ...................................................................................... 39
R
reference markers ................................................................... 20
designating ...................................................................... 31
detection ......................................................................... 52
incorrect detection ........................................................... 55
positioning before gel casting ........................................... 21
positioning before gel scanning ........................................ 22
reference markers area ........................................................... 47
result files
export .............................................................................. 62
location ............................................................................ 56
open ................................................................................ 61
rinse strokes ........................................................................... 46
rinse volume ........................................................................... 46
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Index
IX
S
safety
labels ............................................................................... 16
other warnings ................................................................. 16
precautions ...................................................................... 15
scanner correction ................................................................... 51
selecting spots
automatic ......................................................................... 30
ImageMaster .................................................................... 27
manual ............................................................................ 30
silver staining .......................................................................... 70
Hoefer automatic gel stainer ............................................. 71
PlusOne method .............................................................. 70
SYPRO orange staining ............................................................ 69
SYPRO ruby staining ............................................................... 69
system setup ........................................................................... 40
T
threshold ................................................................................. 52
Typhoon ...........................................................................13, 26
Ettan Spot Picker User Manual 18-1147-61 Edition AA
75
IX
76
Index
Ettan Spot Picker User Manual 18-1147-61 Edition AA
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