Bio-Rad PDQuest 2-D User Manual

ProteomeWorks Plus

™

Spot Cutter System with PDQuest Basic Excision Software

Instruction Manual

Catalog Numbers 165-7064 ProteomeWorks Plus Spot Cutter System 165-7065 ProteomeWorks Plus Spot Cutter with Fluorescent Enclosure

System

For technical service call your local Bio-Rad office or in the U.S. call 1-800-4BIORAD (1-800-424-6723) On the Web at http://www.discover.bio-rad.com

Table of Contents

Section 1 Introduction ......................................................................................1

1.1 General Description ..............................................................................1

1.2 Manual Guidelines ................................................................................1

1.3 System Components ............................................................................1

1.4 Safety Information ................................................................................2

1.5 Caution ................................................................................................3

1.6 Moving the Spot Cutter ........................................................................3

1.7 Computer Recommendations ..............................................................3

1.8 Cutting Excision Sizes ..........................................................................3

1.9 Voltage Specifications ..........................................................................3

1.10 System Description ..............................................................................4

Section 2 Installation ........................................................................................8

2.1 Overview ..............................................................................................8

2.2 Installing PDQuest Software..................................................................8

2.3 Install PCI Card ....................................................................................9

2.4 Spot Cutter Setup ................................................................................9

2.5 Camera Installation ............................................................................10

2.6 Camera Lens Aperture Settings ..........................................................12

2.7 Cable Setup........................................................................................12

2.8 Power ON Startup Sequence ............................................................13

2.9 Spot Cutter Model Number Selection ................................................14

2.10 Camera Focus ....................................................................................15

2.11 Camera Alignment ..............................................................................17

2.11.1 Camera Rotation Alignment ..................................................17

2.11.2 Front/Back Centering of the Camera ....................................18

2.11.3 Lateral Centering of the Camera ............................................18

2.12 Cutting Tip Height ..............................................................................20

2.13 Microtiter Plate (MTP) Calibration ........................................................21

2.14 Wash Well Position Calibration............................................................23

2.15 Fluorescent Enclosure Setup ..............................................................23

Section 3 System Calibration ........................................................................27

3.1 Switch to White Light Imaging ............................................................27

3.2 Prepare Coomassie Stained PVDF Membrane....................................27

3.3 Calibration of X, Y Coordinates ..........................................................28

Section 4 Software Features Description....................................................30

4.1 Spot Cutter Settings ..........................................................................30

Section 5 Spot Excision ..................................................................................33

5.1 Free Gel and Blot Preparation ............................................................33

5.2 Plastic and Glass Backed Gel Preparation..........................................34

5.3 Using PDQuest Basic Excision Tool Software ....................................34

5.3.1 Step 1: Acquire Image ..........................................................35

5.3.2 Step 2: Specify Cuts ..............................................................38

5.3.3 Step 3: Set Cut Options ........................................................40

5.3.4 Step 4: Set Wash Options......................................................41

5.4 Start Spot Excision ............................................................................42

5.4.1 Begin/Resume and Pause......................................................42

5.4.2 Re-cuts in Basic Excision Tool ..............................................42

5.5 Confirm Cuts ......................................................................................46

Section 6 Accessories ....................................................................................47

6.1 Use of Gel Sheets and Gel Positioning ..............................................47

6.2 Common Re-order Parts ....................................................................48

Section 7 Maintenance....................................................................................48

7.1 General Maintenance and Fuses ........................................................48

7.2 Cutting Tip Cleaning ..........................................................................48

7.3 Changing Cutting Tips ........................................................................49

7.4 Set Cutting Tip Height ........................................................................49

7.5 Adjusting the Cutting Carriage ............................................................50

7.6 Ejection Pin ........................................................................................51

Section 8 Troubleshooting..............................................................................52

8.1 Communication Error..........................................................................52

8.2 Image Quality......................................................................................53

8.3 Lights ..............................................................................................53

8.4 Spot Cutting and Pickup ....................................................................54

8.5 Technical Service ................................................................................54

Section 9 Warranty ..........................................................................................55

Appendix A Spot Cutter – Front ........................................................................56

Appendix B Working Surface ............................................................................57

Appendix C Spot Cutter – Back ........................................................................58

Appendix D PDQuest Version 7.0 and 7.1 ......................................................59

Glossary ..................................................................................................................61

System Records ....................................................................................................62

Section 1 Introduction

1.1 General Description

The ProteomeWorks Plus Spot Cutter System is a precision instrument for the accurate location, excision and loading into microtiter plates (MTP) of protein spots after separation on 2-D electrophoresis gels or PVDF membranes. The system has been designed to be both robust in construction and simple to operate.

The ProteomeWorks Spot Cutter with Fluorescent Enclosure is capable of accurately cutting spots from gels and membrane blots with visible stains such as silver and coomassie, and with fluorescent SYPRO Ruby protein stain. The system includes a 620/60 nm bandpass filter that is optimized for fluorescent SYPRO Ruby protein stain.

1.2 Manual Guidelines

If you are installing a ProteomeWorks Plus spot cutter and the ProteomeWorks fluorescent enclosure together, use this instruction manual for the installation and operation of both devices. If you are adding a ProteomeWorks fluorescent enclosure to an existing ProteomeWorks spot cutter or ProteomeWorks Plus spot cutter, then use the Fluorescent Enclosure instruction manual (part number 4006206) supplied with the enclosure.

1.3 System Components

The ProteomeWorks Plus Spot Cutter System is shipped in two boxes under the catalog order number 165-7064. The spot cutter instrument and accessories box is catalog number 165-7063 and the Camera System box is catalog number 165-7070.

The ProteomeWorks Plus Spot Cutter with Fluorescent Enclosure System is shipped in three boxes under the catalog order number 165-7065. The spot cutter instrument and accessories box is catalog number 165-7063, the fluorescent enclosure is catalog number 165-7044, and the Camera System box is catalog number 165-7070.

In addition to the instrument boxes, 1 pack of precut PVDF membrane (162-0255) and 1 L of coomassie stain solution (161-0436) is shipped as part of this order and is used for calibration of the spot cutter.

A focusing / fluorescent imaging target is included for use with the Fluorescent Enclosure and the spot cutter. This target provides focussing lines for setting up the spot cutter camera. It also provides fluorescent squares that can be imaged in the UV light mode. It is suggested that this target be used to test the camera and UV lights before imaging a gel.

1.4 Safety Information

Caution Symbol

Read the manual before using the ProteomeWorks Spot Cutter. For technical assistance, contact your local Bio-Rad or, in the U.S., call technical services at 1-800-4BIORAD (1-800-424-6723).

Definition of Symbols

Caution (refer to instruction manual).

Notice

This instrument is intended for laboratory use only. This product conforms to the "Class A" standards for electromagnetic emissions intended for laboratory equipment applications. It is possible that emissions from this product may interfere with some sensitive appliances when placed nearby or in the same circuit as those appliances. The user should be aware of this potential and take appropriate measures to avoid interference. This Bio-Rad instrument is designed and certified to meet EN-61010-1 safety standards. Operation of this product is subject to the condition that no harmful radio interference is caused and that any interference must be accepted. EN-61010-1 certified products are safe to use when operated in accordance with the instruction manual. This instrument should not be modified in any way. Alteration of this instrument will:

• Void the manufacturer's warranty.

• Void the EN-61010-1 safety certification.

• Create a potential safety hazard.

Bio-Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other than those for which it is intended, or by modifications of the instrument not performed by Bio-Rad or an authorized agent.

For technical assistance, contact your local Bio-Rad or, in the U.S., call technical services at 1-800-4BIORAD (1-800-424-6723).

EN61010-1 is an internationally accepted electrical safety standard for laboratory instruments.

1.5 Caution

It is detrimental to the system to leave the lighting on for more than 10 minutes at a time as it may lead to permanent damage to sections of the system.

1.6 Moving the Spot Cutter

The System should be moved:

• In the upright position.

• By lifting and carrying from the base.

• By placing down in the upright position on a stable and level surface.

Note: DO NOT move the system by lifting the system up by the camera arm.

1.7 Computer Recommendations

Processor 300 MMX or greater

2.1G Hard Drive or greater RAM 128 Mb or greater Screen Resolution 1024 x 768 Communication port 1 serial port is required

Add PCI Interface card (included with spot cutter) 2 Mb Video RAM

Operating system Windows 98, NT, or 2000

1.8 Cutting Excision Sizes

Nominal Tip Size:

1.5 mm internal diameter: Supplied with spot cutter

1.0 mm internal diameter: Supplied with spot cutter

1.9 Voltage Specifications

Nominal input voltage range 100 V to 240 V (50 Hz/60 Hz) Power consumption 220 W

1.10 System Description

Fig. 1. The ProteomeWorks Plus Spot Cutter.

• The Spot Cutter Bench footprint of 50 x 50 cm with a curving camera support arm holding a camera over the platform of the work surface. Under platform lights used for gel imaging, an x,y arm supporting the cutting head mechanism, the gel or PVDF cutting area, a microtiter plate holding area, wash well area containing 4 wash tubes.

• The Camera Cooled CCD camera, 1392 x 1040 imaging pixels, 12 bit ADC

• Working Surface The working surface is shown in Appendix B and consists of two main areas, the cutting surface and the microtiter plate holder / wash wells area. In addition there is also a bubble level which is used to ensure the cutting surface is level.

• Cutting Surface The cutting platform dimensions are 30 x 27 cm. The cutting tip can access an area of 26 cm x 24 cm within this 30 x 27 cm area. The imaging area is approximately 26 x 21 cm. The design of the system allows the ProteomeWorks Plus spot cutter to cut spots from the protein resolving area of any commercially available gel type. The platform area was designed to accommodate the entire gel without the need for trimming the gel to fit the cutting platform. The diagram below shows the orientation of the cutting area within the platform. Gels should be positioned against the right wall of the cutting platform, next to the microtiter plate holder, and centered vertically in the platform.

Fig. 2. Cutting Platform and Cutting Area.

• ProteomeWorks Plus Cutting Mats, catalog number 165-7058. One cutting mat is provided with the system. The cutting mats provide for a flexible surface beneath the gel to allow for complete excision of the gel spots. The mats may become scored by the cutting tip, stained by the gel stains, or become cracked with repeated use over time. This is normal wear and replacement ProteomeWorks Plus Cutting Mats should be ordered from Bio-Rad.

• ProteomeWorks Plus Gel Cutting Sheets, catalog number 165-7018. Gel Cutting Sheets are thin plastic sheets that are positioned between the cutting mat and the gel in the cutting area. This prevents scoring by the cutting tip and extends the life of the cutting mat. The Gel Cutting Sheets can also be used to lift gels out of storage trays to minimize damage to gels from excessive handling. (If a Gel Cutting Sheet will be used during gel excision make sure that the cutting tip height is adjusted accordingly. See Section 7.4 "Set Cutting Tip Height".)

• Gel-holding bars Two stainless steel Gel-holding Bars are included in the accessory box with the spot cutter. These gel holding bars are used to immobilize the Coomassie stained PVDF membrane during the X, Y calibration procedure, as well as for general gel excision. Correct placement of the gel holding bars on the gel prevents gel movement during the gel excision process. Position the bars resting on the outer edge of the gel or membrane. It is best to position them to the left side and bottom of the gel so they are out of the cutting tip travel path.

Fluorescent Enclosure

• The cabinet

• The door

• Quick release hinges

• Safety interlock switch

• Base mat

• UV-B lights

• 620 / 60 nm band pass filter

These parts are described in Figure 1.

Fig. 3. Fluorescent Enclosure with spot cutter.

Software Description

The ProteomeWorks Plus Spot Cutter requires PDQuest version 7.1.1 or greater for operation. The ProteomeWorks Plus Spot Cutter is shipped with a Discovery Series install CD which includes PDQuest software but does not include a License for PDQuest software. PDQuest software runs the spot cutter in two modes.

• The Basic Excision Tool provides the capability for imaging, calibrating, visually selecting spots to cut, cutting them, and tracking the microtiter plate well they were deposited in. It does not include any image analysis capability.

• With a separate purchase of a license for full use of PDQuest image analysis software, additional functionality is available. In addition to the use of the outstanding 2-D image analysis features of PDQuest software, the full license provides the ability to use PDQuest analysis sets to run spot excision, and all spot number tracking is kept within the PDQuest matchset database. The manual spot selection of the Basic Excision tool is still available as a menu option in the licensed version.

Fig. 4. Example PDQuest Basic Excision software screen.

The Discovery Series CD contains two separate software components that work together to operate the ProteomeWorks Plus Spot Cutter: PDQuest operating software, and RS Config camera driver software. Spot Cutter operation requires that all components be installed properly. After installing the software, the Spot Cutter will be operated using PDQuest exclusively. The other software component serves only to support the use of PDQuest with the Spot Cutter. PDQuest and the supporting software are compatible with Windows 98, Windows NT 4.0, and Windows

2000.

Section 2 Installation

2.1 Overview of Installation Process

The following is an overview / checklist of the process for installing and calibrating the spot cutter. Use this to review that all the steps have been completed.

1. Install PDQuest software

2. Install PCI card in computer

3. Setup spot cutter and camera

4. Cable set-up

5. Power on sequence

6. Open Basic Excision Tool

7. Select Spot Cutter model

8. Camera focus

9. Camera alignment

10. Set cutting tip height

11. Microtiter plate calibration

12. Wash tube calibration

13. Install Fluorescent Enclosure (optional)

14. Switch to white light setting

15. System calibration

2.2 Installing PDQuest Software

The following procedure applies to new installations of PDQuest version 7.2 and higher. If you are installing PDQuest version 7.0 or 7.1, see Appendix D.

Important Note: Install PDQuest with the "Spot Cutter Drivers" BEFORE physically installing the PCI card hardware in the computer.

Installation Procedure:

1. Insert The Discovery Series /PDQuest in the CD-ROM. The disk will start up with the

"Discovery Series" Window.

2. Select the PDQuest Installer.

3. Click "Next" on the Welcome Screen.

4. Accept the License Agreement.

5. Click "Next" on the Readme Screen.

6. Enter Full Name and Organization and click "Next".

7. Select installation directory and click "Next".

8. Enable the “Spot Cutter drivers” as shown in the screen below.

9. Click "Next" and follow the rest of the on-screen instructions to complete the installation.

10. When the installation is complete, shut down the computer to complete "power off".

Note: Installation of PDQuest software includes the Basic Excision Tool for operation of the ProteomeWorks Plus spot cutter. See Section 4 for a description of spot cutter operation through the Basic Excision Tool. The full analysis features of PDQuest software are available through the purchase of a software license.

2.3 Install PCI Card

1. Install the PCI card in an open PCI slot on the computer mother-board.

2. Close the computer cabinet.

3. Start up the computer and let it boot up to the Windows operating system. Windows

will find the new hardware and present a message to install new hardware. Click on the option that installs the new hardware. It will automatically use the Spot Cutter Driver, you do not need to direct it to the location.

4. All the software needed to operate the spot cutter is now installed. Proceed with the

next step in the hardware setup.

2.4 Spot Cutter Setup

Note: the ProteomeWorks spot cutter Video CD has a video tutorial that goes through the

process of the camera setup and alignment. Request the Video CD, catalog number 165-7046.

• Remove the Spot Cutter instrument and accessories from the shipping container. Lift the

instrument by the base, do not lift the instrument by the camera arm.

• Remove the metal packing bars from the feet by loosening the crescent bolts on the spot cutter feet. A flat crescent wrench is supplied in the accessories box for this purpose. Place the instrument on a flat surface. If you are also installing the ProteomeWorks Fluorescent Enclosure, place the spot cutter on the black base mat from the Fluorescent Enclosure packaging. Add the fluorescent enclosure after the camera installation is completed. This will make the camera installation easier.

• Carefully remove the cutting tip protector and tape.

• Remove the two "L" Shaped brackets from the system X, Y arm, there is one bracket located at each end of the X, Y arm. They are secured with 2.5 mm hex screws.

• Move the X, Y arm forward to access and remove the third L bracket located on the back of the X, Y arm and the cutting head assembly. Do not move the X, Y arm when the instrument is powered on.

• Level the instrument to the bubble level on the platform surface using the leveling feet.

2.5 Camera Installation

• Remove the camera mount and mounting screws from the Camera System box (catalog number 165-7070).

• The camera mount is bolted to the end of the camera arm. Position the camera mount on the camera arm of the spot cutter and use the 6 mm hex wrench from the accessories box to secure the camera mount onto the camera arm with the large hex screw.

• Tighten the screw to snug, but do not overtighten. Some movement is needed for the camera alignment procedure.

Fig. 5. Camera and mount.

• Remove the camera from the Camera System box and remove the blue protective film from the front of the camera.

• Remove the 8 mm lens from the Camera System box.

• Use the red handled 2 mm hex driver in the accessories box to tighten the 2 mm hex screw in the silver locking collar of the lens (if it is loose) before mounting it on the camera.

• Screw the lens into the C-mount fitting on the camera until snug.

• Install the bandpass filter on the lens using the following steps, even if you are not installing the Fluorescent Enclosure.

1. The bandpass filter is shipped in the Camera System box 165-7070. It includes a

filter holder, a 620/60 nm bandpass filter, and a 0.9 mm hex wrench.

Fig. 6. Lens and Bandpass filter set.

2. Screw the filter into the filter holder. The threads are very fine, so be careful not to

cross the threads.

Fig. 7. Lens and filter on camera.

3. To attach the filter holder to the lens of the camera, slide the filter holder up over the

lens until stops. Using the 0.9 mm hex wrench, gently tighten the setscrews on the side of the filter holder.

Note: Bandpass Filter The bandpass filter is used for both the white light (visible stain) and UV light (fluorescent stain) modes. Once it is installed, it is not taken off the lens. The only adjustment to the camera between the two light modes is a change in the aperture from the nearly closed (f-stop 11–16) position for white light, to the open (f-stop 1.4–2.8) position for UV lights.

• Attach the camera to the camera arm by placing the camera against the ridge of the camera mount. Secure the camera to the mount with the camera screw through the slot in the camera mount.

2.6 Camera Lens Aperture Settings

The aperture setting on the lens is a black ring on the lens with a small black thumbscrew attached. There are aperture settings marked on two sides of the lens. The white mark on the ring indicates the aperture setting. F-stop setting 1.4 is maximum open for the lens; f-stop 11-16 is the maximum closed for the lens (16 is not marked but is past 11). See Figures 8 & 9 for open and closed aperture settings. To adjust the aperture setting, loosen the thumbscrew, move the ring to the desired setting and gently tighten the thumbscrew. Tightening the thumbscrew is optional; the aperture setting does not drift but can be accidentally changed if it is touched.

Fig 8. Aperture at f-stop 1.4 for UV light mode. Fig. 9. Aperture at f-stop 16 for white light mode.

2.7 Cable Setup

• Connect the PC cable. Connect the 9-pin connector to the Com 1 serial port of the computer and the 25 pin connector to the system communications interface located in the back panel of the Spot Cutter.

• Connect the AC power cord to the Spot Cutter instrument.

• Connect the camera power and communications cable to the PCI interface port on the

PC and the connector on the camera top. Secure the cable through the clips on the back of the camera arm. See Section 2.2 for correct sequence of camera driver &

PCI card installation.

Fig. 10. Rear panel connections.

2.8 Power "ON' Startup Sequence

Before powering on and opening the software, make sure that all the cables have been correctly installed.

It is important for the correct sequence of devices to be powered on. The computer must be turned on and fully booted up before the spot cutter is turned on. If the spot cutter is on first, or if the computer is re-booted while the spot cutter is on, several commands will not be communicated. The symptoms are: lights not turning on, and cutting tip not spinning with cut. When powering up the spot cutter system (including computer) use the following sequence:

1. Turn on the computer and let it boot up fully to its operating system. Do not open PDQuest yet.

3. Turn on the spot cutter (switch the front black I/O switch to I), wait a second, then push the yellow "Start/Pause" button. Let the spot cutter run through its home position routine until it returns to the back right position (park position) and stops moving.

4. Turn on the camera with the black rocker switch on top of the camera. The green light on the top indicates the power is on.

5. Open PDQuest and go to either Basic Excision Tool, or Excision Gel Selection.

2.9 Spot Cutter Model Number Selection

The ProteomeWorks Plus spot cutter is the second generation spot cutter from Bio-Rad. The first model was the ProteomeWorks spot cutter. These different models can be identified by the name plate on the front of the instrument, reading either ProteomeWorks Plus spot cutter or ProteomeWorks spot cutter. The physical dimensions of the platform gel holding area are different.

ProteomeWorks Plus spot cutter = 30 x 27 cm ProteomeWorks spot cutter = 25 x 25 cm

PDQuest version 7.1.1 and higher, support both models of spot cutters. When there is no calibration information on the computer running a spot cutter, the first time that the spot cutter communication is opened through PDQuest, the Spot Cutter "settings" panel will open. You will be prompted to indicate which model of spot cutter you are using. You must select one of the models before any of the operational screens will be active.

Fig. 11. Settings panel model selection at initial start up.

The ProteomeWorks spot cutter (25 cm) model does not support the "Backed Gel" cutting option. The software functions related to Backed Gels will be greyed out and unavailable for use.

Fig. 12. ProteomeWorks spot cutter Settings panel

If you select the Pr

oteomeWorks Plus spot cutter model, set the “Crop left margin” to 50. Click

“Done” to save the changes and close the window.

The rest of the functions and set up are the same between the two models of spot cutter.

2.10 Camera Focus

Focus using the following steps.

• Loosen the 2mm hex screw in the silver lock collar of the lens with the red handled 2 mm hex driver.

• Loosen the black aperture thumbscrew

• Set the aperture to the open setting of f-stop 1.4

• On the back portion of the spot cutter instrument, behind the cutting platform, is a black toggle switch. This toggle switches the power to the white lights on or off. It is not the mains power switch. Switch the toggle to the lights off position. Check that the lights are off by pushing the green "Lights on" button on the lower front right side of the spot cutter. If the platform lights come on, switch the toggle switch to the other position.

Note: Toggle Switch for White or UV Lights

To switch from one light mode to the other, the black switch on the back of the ballast box on the spot cutter is toggled from one position to the other.

Fig. 13. Toggle switch on back of spot cutter.

• Remove the cutting mat, and any other materials, from the cutting platform.

• Click the "Focus Cutter Camera" icon from the Basic Excision Tool window.

• Click the "Auto-expose" button. The software will reset the exposure time of the images. Click "OK" when "Auto-expose completed". Do not click "Stop". The focus mode window will continue on the screen and continue acquiring images.

• Put the cutting mat and the Bio-Rad Focusing Target or the Calibration Template on the cutting mat for focusing.

• You will see the center portion of the platform in continuous acquire mode in the window.

• Center the Bio-Rad Focusing Target so that you can see the fine lines that are close together. Focusing the lens will resolve these lines from one another and sharply focus the camera.

• To focus, physically turn the lens and watch both the image in the window and the "Focus levels" box. The image should resolve the lines and bring the image into sharp focus. The "Focus levels" box is a gauge of the gray scale across the image. When the focus is at the best point, the "Focus levels" number should reach a bottom plateau. This is meant as a guide rather than a definite number point.

• When the focus is set, lock the camera lens in place by tightening the silver lock collar. Hold the lens in place with one hand and tighten the 2 mm hex screw in the silver lock collar.

• Click "Stop" on the "Focus cutter camera" window before closing the window.

• Keep the light setting at the same "lights off" position for the camera alignment steps.

Any time the camera focus is changed, the spot cutter must be re-calibrated. The aperture can be changed without re-calibrating.

2.11 Camera Alignment

(Tools needed from the accessory box: the 6 mm and the 3/16 inch mm hex driver)

2.11.1 Camera Rotation Alignment

• Place the Calibration Template (grid patter on clear plastic) on the cutting mat in the cutting platform. Remove the protective film coating from both sides of the template.

• Click on the Focus Cutter Camera icon in the Basic Excision Tool screen. If the image is over exposed, check the light and aperture settings. If the settings are correct, take the cutting mat off the platform, click on "Auto-expose" and then click "OK" when "Autoexpose completed". Place the cutting mat and Calibration Template back on the cutting platform and proceed.

• Loosen the 6 mm camera mount screw slightly to be able to rotate the camera. The screw is located on the under side of the curved arm supporting the camera.

• Rotate the camera mount until the black and red lines are parallel. Note, the lines will not overlay exactly. See Figure 14 for an example

• Tighten the bottom mounting screw while holding the camera in the correct position. The lines must remain parallel.

Fig. 14. Correct rotation alignment.

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