Imaging Systems Criterion Stain Free™ Imaging System Instruction Manual
Criterion Stain Free System Instruction Manual Bio-Rad Laboratories, Inc.
Criterion Stain FreeTM System Copyright ©2008 Bio-Rad Laboratories, Inc. Reproduction in any form, either print or electronic, is prohibited without written permission of Bio-Rad Laboratories, Inc. Criterion Stain Free Imager, Criterion Stain Free Gels, and Image Lab™ are trademarks belonging to Bio-Rad Laboratories, Inc. Other brands or product names are trademarks of their respective holders. NOTICE The Criterion Stain Free™ Imaging System is intended for laboratory use only.
Safety and Regulatory Compliance The Criterion Stain Free™ Imaging System is intended for laboratory use only. This instrument is meant for use only by specialized personnel who know the health risks associated with the reagents normally used with this instrument. The UV light source is computer controlled and proper interlocks are implemented to avoid the user’s accidental exposure to UV radiation.
Criterion Stain FreeTM System | Safety and Regulatory Compliance For easy customer access, the serial number information is in two locations on your instrument; on the back panel, and inside the front door. Instrument Safety Warnings Notes, cautions and warnings are used to highlight certain operating procedures and recommendations. The table below describes how each is used in this document. Table 1.
The technical specifications of the Criterion Stain Free Imager are in the table below: Table 2. Technical Specifications Hardware Specifications Image Area 13.9 x 9.4 cm Excitation Source Trans UV 302 Detector CCD Image Resolution (Horizontal x Vertical) 1392 x 940 pixels Image Pixel Size (in Microns) 100 x 100 um Dynamic Range 3.0 Orders Pixel Density (gray levels) 4096 Flat Fielding Yes Instrument Size 43 x 28 x 38 cm Instrument Weight 7.
Criterion Stain FreeTM System | Safety and Regulatory Compliance CAUTIONS CAUTION: With the exception of cleaning or replacing light bulbs, refer all servicing to qualified Bio-Rad personnel or their agents. If you experience technical difficulties with the instrument, contact Bio-Rad to schedule service. The instrument should not be modified or altered in any way. Alteration voids the manufacturer’s warranty and may create a potential safety hazard for the user.
Table of Contents Safety Use Specifications and Compliance . . . . . . . . . . . . . . . . . . . . . .iii Instrument Safety Warnings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv Connector Descriptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Cautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi Warnings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi Chapter 1.
Criterion Stain FreeTM System | Table of Contents Chapter 3. Criterion Stain Free Gels . . . . . . . . . . . . . . . . . . . . . 15 Available Criterion Stain Free Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Criterion Comb Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Gel Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Setup and Basic Operation . . . . . . . . . . . . . . . . . . . . . . . .
Chapter 5. Designing Protocols . . . . . . . . . . . . . . . . . . . . . . . . 33 Running the Default Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Creating and Running Custom Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Protocol Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Protocol Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Protocol Steps . . . . . . . . . . . . . . . .
Criterion Stain FreeTM System | Table of Contents Image Colors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 3-D Projection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Image Info . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Image Details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Analysis Settings . . . . . . . . . . . . . . . . .
4. Molecular Weight Analysis Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Molecular Weight Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Standard Lanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Regression Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 5. Annotation Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Add Annotations . . . . . . . . . . . . . . . . . . . . .
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Criterion Stain FreeTM System 1 Criterion Stain Free System Overview This manual describes the Criterion Stain Free™ System, which consists of the Criterion Stain Free™ Imager, Criterion Stain Free™ Gels, and Image Lab™ software. The Criterion Stain Free System is a reproducible and fast label-free SDS-PAGE/Native PAGE system that eliminates time-consuming staining and destaining steps.
Criterion Stain FreeTM System | Criterion Stain Free System Overview Applications Image Lab software has been designed to perform automated protocols for routine gel imaging documentation and analysis. Gel Documentation Gel electrophoresis is a common way to separate, identify, and purify proteins. The Criterion Stain Free System allows users to image and print gels for documentation in laboratory notebooks, or to export images for publication or presentations.
Overview of the Imaging Process Overview of the Imaging Process There are two ways to image your gel on the Criterion Stain Free Imager. Image Lab must be open on your PC, and the Imager must be on. You can run the default protocol included with Image Lab, which activates and images the gel with one touch of a button, or customize a protocol based upon your exact requirements. To run a default protocol: 1. Open the Imager door and remove the sample tray. 2.
Criterion Stain FreeTM System | Criterion Stain Free System Overview Criterion Stain Free Imaging System The Criterion Stain Free Imaging System is a dedicated low-noise detection camera and UV transilluminator-based system with a small footprint. Designed for economy, automation, and ease of use, the user interface consists of a sample tray and a one-touch button to image your gel using either a default or custom protocol.
Image Lab Software Image Lab Software Minimum configurations for Image Lab software: • Windows XP or Vista operating system with Pentium-4 or later processor and 1 GB RAM • Macintosh OSX 10.5 or later operating system, Intel processor and 1 GB RAM. Refer to Chapters 4 through 9 for instructions on using Image Lab software. Setting Up the System Installing the Image Lab Software MACINTOSH 1. Place the Image Lab software CD in your CD-ROM drive. 2.
Criterion Stain FreeTM System | Criterion Stain Free System Overview 2. When the Image Lab software icon appears on your desktop, doubleclick to open the software. Connecting the Imager to the Computer 1. Install the Image Lab software before connecting the Criterion Stain Free Imager to a PC computer. 2. Connect the Imager to the PC, using the USB cable provided with the system. 3. Connect the Criterion Stain Free Imager to AC power, using the provided power cord.
Setting Up the System IF YOUR COMPUTER IS RUNNING WINDOWS XP: After the power is turned on, the PC will automatically recognize the Imager, and the software will prompt you to install the drivers. The screen below appears: 5. Select “No, not this time”, and then, “Next”.
Criterion Stain FreeTM System | Criterion Stain Free System Overview 6. “Install the software automatically” is selected by default. Click on “Next”. The screen below appears: 7. Click on “Continue Anyway”. Windows will install the driver. 8. Click on the “Finish” button when it appears. A message appears briefly on the lower right corner of the screen indicating “Your new hardware is ready to use”. NOTE: Windows XP may prompt you to go through the driver installation a second time.
Setting Up the System IF YOUR COMPUTER IS RUNNING VISTA: After the power is turned on, the PC will automatically recognize the Imager, and the software will prompt you to install the drivers. This screen appears: 1. Click “Locate and install driver software”. Vista finds the drivers automatically. 2. You may see this screen. If you do, click “Install this driver software anyway”. The drivers will be installed. 3. Click on the “Finish” button when it appears.
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Criterion Stain FreeTM System 2 Criterion Stain Free Imager The Criterion Stain Free™ Imager is a quantitative imaging system for capturing high resolution digital images of Bio-Rad Criterion Stain Free™ gels. Using low noise CCD technology, exceptionally uniform UV-B excitation, and a highly efficient optical design, the Criterion Stain Free Imager offers researchers the sensitivity, uniformity, flexibility, and dynamic range required for the analysis of electrophoretically separated proteins.
Criterion Stain FreeTM System | Criterion Stain Free Imager You can watch the first image of your gel progress through the development process, obtaining usable data within 2.5 minutes. You can choose to develop the gel for up to 5 minutes, to ensure your sample is fully developed. The level of gel development is user controllable, allowing for data that suits your needs.
Mechanical Description Mechanical Description The Criterion Stain Free Imager consists of the following main hardware components: • Light-tight enclosure, which contains a UV transilluminator • CCD camera • Low distortion lens • Removable sample tray with UV-B filter glass • Electronics for controlling the UV lights and camera WARNING: Do not remove the instrument cover when power is supplied to the instrument, or defeat the UV safety interlock.
Criterion Stain FreeTM System | Criterion Stain Free Imager 4. On the left side of the instrument there is a single screw holding the lamp assembly in place. Remove and save this screw. 5. Carefully slide out the lamp assembly. Although only one lamp bulb may be out, it is recommended that all lamps be replaced to ensure even illumination and reduce the need to access the lamps again. 6. Replace all lamps with UV-B fluorescent lamps (Bio-Rad PN 900-0217).
Criterion Stain FreeTM System 3 Criterion Stain Free Gels Because of Criterion Stain Free™ Gels’ unique composition, allowing gel imaging without staining, you must use them with the Criterion Stain Free™ Imager. The following table details Bio-Rad Stain Free Gels.
Criterion Stain FreeTM System | Criterion Stain Free Gels Gel Specifications Gel material Polyacrylamide Gel dimensions 8.7 x 13.3 cm Gel thickness 1.0 mm Resolving gel height 6.5 cm Cassette dimensions 10.6 x 15.
Setup and Basic Operation 7. Fill each half of the lower buffer tank with 400 ml of running buffer to the marked fill line. 8. Place the lid on the tank, aligning the color-coded banana plugs and jacks. See “Running Conditions” (page 21) for power conditions. Opening Criterion Cassettes and Removing the Gel 1. After electrophoresis is complete, turn off the power supply and disconnect the electrical leads. 2. Remove the lid from the tank, and remove the Criterion gel from the cell.
Criterion Stain FreeTM System | Criterion Stain Free Gels SDS-PAGE Criterion Tris-HCl gels provide a versatile system for the separation of proteins by molecular weight (SDS-PAGE conditions) or charge-to-mass ratio (Native conditions). (See page 20 for Native PAGE applications and protocols.) This versatility is possible because Criterion Tris-HCl gels are made without SDS, allowing the sample buffer and running buffer to determine the separation mechanism.
SDS-PAGE Criterion Tris-HCI Gel Separation Guide Tris-HCl gels are available in a wide selection of single percentages and gradients for the separation of proteins by SDS-PAGE. Tris-HCI Gels Optimal Separation 10% 30-150 kD 4-20% gradient 10-200 kD SDS-PAGE Buffers Running buffer, 1x 25 mM tris 192 mM glycine 0.1 SDS DO NOT ADJUST pH Sample buffer 62.5 mM Tris-HCI, pH 6.8 2% SDS 25% glycerol 0.
Criterion Stain FreeTM System | Criterion Stain Free Gels Native PAGE Criterion Tris-HCl gels are made without SDS, allowing separation of proteins in their native conformation. The nonreducing and nondenaturing environment of Native PAGE allows the detection of biological activity and can improve antibody detection. Native PAGE can also be used to resolve multiple protein bands where molecular mass separation by SDS-PAGE would reveal only one.
Native PAGE Native PAGE Buffers Running buffer, 1x 25 mM Tris 192 mM glycine DO NOT ADJUST pH Sample buffer 62.5 mM Tris-HCI, pH 6.8 25% glycerol 0.01% Bromophenol Blue Sample Preparation Determine the desired protein concentration and load volume of your sample based on the detection method used. Sample preparation for Native PAGE applications requires special consideration. In the absence of SDS, the net charge of a polypeptide will be determined by the pH of the sample buffer.
Criterion Stain FreeTM System | Criterion Stain Free Gels Electrophoresis Troubleshooting Improper storage of Criterion gels can produce numerous artifacts. Criterion gels should be stored flat at 4ºC. Avoid freezing or prolonged storage above 4ºC. If you suspect your gels have been stored improperly, DO NOT USE THEM.
Electrophoresis Supplies Electrophoresis Supplies Criterion Gel Accessories Accessories Catalog # Criterion Sample Loading Guide, 12+2 well, 1 165-6006 Criterion Sample Loading Guide, 18-well, 1 165-6007 Criterion Sample Loading Guide, 26-well, 1 165-6008 Buffers Premixed Running Buffers Catalog # 10x Tris/Glycine/SDS, 1 L 161-0732 10x Tris/Glycine/SDS, 5 L 161-0772 10x Tris/Glycine, 1 L 161-0734 10x Tris/Glycine, 5 L 161-0771 Premixed Sample Buffers Catalog # Laemmli Sample Buffer, 30
Criterion Stain FreeTM System | Criterion Stain Free Gels Individual Reagents Tris, 1 kg 161-0719 Glycine, 1 kg 161-0718 SDS, 100 g 161-0301 2-Mercaptoethanol, 25 ml 161-0710 Dithiothreitol (DTT) , 5 g 161-0611 Bromophenol Blue, 10 g 161-0404 Protein Standards Bio-Rad Precision Protein Plus Unstained Standards, 1ml 161-0363 Bio-Rad SDS-PAGE Standards, broad range, 200 μl 161-0317 Bio-Rad SDS-Page Standards, low range, 200 μl 161-0304 Bio-Rad SDS-PAGE Standards, high range, 200 μl 161-030
Criterion Stain FreeTM System 4 Image Lab Software Overview Image Lab™ image acquisition and analysis software works with the Criterion Stain Free™ Imager and Criterion Stain Free™ Gels to create a reproducible, automated and time-saving system for running protocols on electrophoresis gels, without time-consuming staining and destaining steps. Researchers can run a default protocol or easily design their own. The data can be viewed, modified, and reported using Image Lab.
Criterion Stain FreeTM System | Image Lab Software Overview Software Overview The following illustration shows the Image Lab main window. The paragraphs below describe the major software elements. Menu Items Toolbar Quick Start Analysis Tool Box Desktop Status Bar Main Window There is one main window used in Image Lab. Results Files, protocols, and dialog boxes presenting choices all open on the Desktop.
Software Overview Menu Items The following section describes all of the choices under the Menu Items; File, Edit, View, Window, and Help. Many of these functions are also available in other locations, such as the Toolbar or the Quick Start Guide. FILE File > New Protocol allows the user to Create a new Protocol through one wizard which contains all of the necessary steps and choices. See Chapter 5, “Designing Protocols” for detailed instructions. The Protocol can also be altered, and stored for reuse.
Criterion Stain FreeTM System | Image Lab Software Overview File > Image Info shows information about the individual gels, such as acquisition date, the data range, and acquisition details, such as the activation time used. There are three tabs (Image Details, Analysis Settings, and Notes) which reveal specific information. The user can enter findings into the Notes tab. File > Page Setup contains print controls, such as orientation (landscape or portrait), margins, printer used and paper size.
Software Overview • The Colors tab allows you to choose colors for the graphic elements in your gels, such as Lane Fame, Lane, Band, Band Attribute and MW Legend. This functionality ensures that these elements will be visible, whatever colors your gels are. VIEW View > Image Overview opens a small window which shows your entire gel image, and a red rectangle outlining the area visible in the larger main window. This is useful when you are zoomed into a small section of your image.
Criterion Stain FreeTM System | Image Lab Software Overview Toolbar Many of the tools available elsewhere in the software can be chosen directly by clicking on these icons. The tools for Viewing Results Data are shown below.These tools are described in detail in Chapter 6, “Viewing Results” on page 43. Tools for Viewing Results Data Analysis Tool Box The Auto Analysis button quickly analyzes images which have not been analyzed previously. The rest of these tools customize the analyzed data.
Software Overview Status Bar The status bar in the lower right-hand corner of the main window shows X and Y values for the cursor position on a results file. The INT (intensity) values show a numerical value for the image intensity at the mouse position. The maximum data range is 0 and 4095, but will vary according to the values contained within your image. See “Generating Reports” on page 65 for information about the options available when you generate your report.
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Criterion Stain FreeTM System 5 Designing Protocols Image Lab™ software can be used to run a simple default protocol, or to design custom protocols with a wide variety of settings. Running the Default Protocol Run the Default Protocol simply by clicking the Run Default Protocol button on the Quick Start Guide, or by choosing File > Default Protocol > Run from the menu bar. A third option is pressing the green Run button on the Imager.
Criterion Stain FreeTM System | Designing Protocols Creating and Running Custom Protocols You start your new protocol by choosing the New Protocol box on the Quick Start Guide, or by choosing New Protocol from the toolbar or menu bar. Custom Protocols involve choosing many parameter settings. PROTOCOL SUMMARY The Protocol Summary screen (shown above) displays first, showing the current parameters.
Creating and Running Custom Protocols Acquire Image STEP 1. ACTIVATE GEL ACTIVATION OPTIONS Visualizing the proteins requires activation of the gel. Exposure of the Stain Free gel proteins to ultraviolet light causes a Stain Free gel component to react with the gel proteins. There are two activation time options, one that runs for 2.5 minutes and one that runs for 5 minutes. This choice is based upon your sample and the purpose of your experiment: • Use 2.
Criterion Stain FreeTM System | Designing Protocols STEP 2. IMAGE GEL EXPOSURE TIME Image Lab will acquire an image using the optimal exposure time, if you select the Auto button. This option will ensure the best use of the dynamic range. Use a longer exposure time to visualize very faint bands. That may cause saturation (overexposure) of more prominent bands, so they cannot be quantified. Exposure time can range from 0.001 to 10 seconds.
Creating and Running Custom Protocols Analyze Image STEP 3. DETECT LANES AND BANDS To perform any analysis on the gel, Image Lab needs to detect lanes and bands on the gel image. The lanes are detected automatically. You have the following choices to customize band detection: LOW BAND DETECTION SENSITIVITY This setting will set detection at a low level, for images with more prominent bands. Faint bands will not be detected with this setting.
Criterion Stain FreeTM System | Designing Protocols STEP 4. ANALYZE MOLECULAR WEIGHT If you want to estimate the molecular weight of the bands in your gel, use this step to enter the standards you are using, and also to specify the lane(s) in which the standards are placed. You analyze molecular weight automatically by clicking Protocol Step 4. “Analyze Molecular Weight” shown above.
Creating and Running Custom Protocols EDIT BUTTON The Edit button opens the Manage Standards dialog box, which allows you to add other third-party standard samples, and to edit your list. STANDARD LANES You can choose which lane will display your Standards, by typing lane numbers, or the words First and Last, in the Standard Lanes field. The format is xx, xx, xx, ... where xx is the lane number.
Criterion Stain FreeTM System | Designing Protocols If you do not have enough data points for the selected method, the molecular weight of the unknown bands will not be calculated. Which method to select depends on the gel type: • 4-20% gradient gels: The Linear (semi-log) regression method works well for these gels, since the mobility of the bands is linear to the log of their molecular weight. As an alternative, the Point-toPoint (semi-log) method can be used, if the r2 value is not good enough.
Creating and Running Custom Protocols Configure and Print Report This menu presents choices about what you want included in an image report. For more complete information, see Chapter 8, “Generating Reports”. STEP 5. PRINT REPORT CUSTOM REPORT TITLE You can provide a custom report title for your Report. INCLUDE GEL IMAGE You can choose whether to include your gel image in the report, and whether lanes appear over the bands.
Criterion Stain FreeTM System | Designing Protocols INCLUDE ANALYSIS TABLE This dialog allows you to choose the parts of the analysis table that are included in your image report. Under Lanes to Show, you can choose all or just some lanes. The Lane Profile can be included in your report, or omitted. Under Columns to Show, you can include the following information. Each type of information is defined below: • Band Number - Each band is numbered from top to bottom, within each lane frame.
Criterion Stain FreeTM System 6 Viewing Results Once your gel has been imaged, the results file appears on the desktop. There are many controls to optimize viewing your results files. You can make use of as many views as you need, and then print your image report. See “Generating Reports” on page 65 to choose which information to include in your Image Reports.
Criterion Stain FreeTM System | Viewing Results Displaying Gel Images Notice the display tool bar icons above the gel image. Each of these tools are described below. Display Gel Options ANNOTATIONS You have a choice whether or not to show text and arrow annotations. LANES AND BANDS You can turn lane frames, lanes, bands, lane labels and molecular weight legends on or off. BAND ATTRIBUTES You can show the following attributes for selected lanes, or all lanes.
Displaying Gel Images Zoom Tools The zoom tools resize your gel image. Click on the magnifying glass with the plus to make your image larger; the minus to make your image smaller. You can also zoom with the right mouse button. Click and drag the right mouse button to marquee an area you want magnified. Click the right mouse button again to return to the original view. Fit in Window Whatever the size of your results file, clicking this button will fit it into the window.
Criterion Stain FreeTM System | Viewing Results Image Transform Use the Image Transform dialog box to adjust image brightness and contrast, optimizing the image display. The minimum to maximum range will vary, according to the light and dark values present in your image. NOTE: These adjustments do not change the data, only the way the data is displayed. The human eye cannot see as much range as is contained in the image.
Displaying Gel Images • • Highlight saturated pixels - when this checkbox is selected, areas of the image with saturated signal intensity are highlighted in red Linear or Logarithmic histogram - click the log checkbox if your image data is in a narrow range, to allow for finer adjustments Image Colors This dialog allows you to choose a colormap for your image results file. Its can be useful to view the image with a different color scheme, making it easier to see all of the elements in the image.
Criterion Stain FreeTM System | Viewing Results 3-D Projection Clicking the 3-D icon transforms your gel image to a solid dimensional model spinning in space with x, y and z dimensions. Relative heights of data points can be accentuated or diminished by pulling the slider to the right or left. Image Info The Image Info dialog box displays three information tabs about your image.
Displaying Gel Images IMAGE DETAILS Acquisition and image Information is displayed in this dialog box. ANALYSIS SETTINGS The settings that were used when the gel was analyzed (Band Detection and whether Molecular Weight Analysis was performed) are displayed here. NOTES You can add notes, make custom labels for each of the lanes, point out the type of samples used, or anything else you want to point out about the results.
Criterion Stain FreeTM System | Viewing Results Displaying Results Data Results data associated with the gel images can be visualized several different ways; as an Analysis Table, a Lane Profile, a Standard Curve, or in a Report. Buttons to toggle these views on and off are located in the main toolbar. All of them can be viewed at one time. Each are described below. Analysis Table Options Clicking on the Analysis Table button opens a tabular display of the data extracted from the Results File.
Displaying Results Data DISPLAY DATA OPTIONS There are three tabs in this dialog box: Measurements, Display and Export. Measurements - Choose the measurements you want displayed by clicking the arrow buttons to move them from the Displayed field to the Not Displayed field. Default display settings - “Move selected lane to top” is on or off, depending on whether the checkbox is on or off.
Criterion Stain FreeTM System | Viewing Results Exporting an Analysis Table The next three buttons offer several ways of exporting an Analysis Table, depending on how the table will be presented. See Chapter 9 for complete information on exporting your files. COPY ANALYSIS TABLE TO THE CLIPBOARD Click this button to copy the Analysis Table to your clipboard, in order to copy your Analysis Table to word processing or presentation applications.
Displaying Results Data Lane Profile Subtracted Background Rotated selected lane The Lane Profile shows you a cross section of the selected lane, rotated to 90 degrees. You can select other lanes by clicking on them while the Lane Profile window is open. The Lane Profile shows the subtracted background under the blue line, and the area used for quantification in faint green dots under the red line. You can change how the bands are identified by choosing from the options in the drop down list.
Criterion Stain FreeTM System | Viewing Results Standard Curve This dialog displays the best fit curve for your defined standards and the bands relative to this curve, for the lane you have selected in the image. Standards are displayed in green, and unknown bands are in red. You can change the molecular weight display on the Y axis between linear and log scale, by clicking the Log MW box at the upper left.
Criterion Stain Free System 7 Editing Results The Analysis Tool Box tools are enabled once a results file is opened and in focus. An active or “in focus” window has a darker blue menu bar on a Windows PC. On a Mac, the window control icons display more brightly when a window is active. This distinction allows you to edit one of many open results files on your desktop.
Criterion Stain Free System | Editing Results DETECTION SETTINGS You have the following choices for band detection: Low Band Detection Sensitivity - This setting will set detection at a low level, for images with more prominent bands. Faint bands will not be detected with this setting. High Band Detection Sensitivity - This setting will set detection at a higher level, for images that are more faint. Extraneous bands can be removed later, using the Band Tools in the Analysis Tool Box. See “3.
Analysis Toolbox MOLECULAR WEIGHT ANALYSIS SETTINGS Molecular Weight Standard - Run any of the four Bio-Rad standards, or third-party standards, which you have placed in your standards list. See “Molecular Weight Standards” on page 38 for full information. Standard Lanes - Choose or change the lanes in which the standards are placed. Regression Method - You have a choice of seven regression methods. For complete information, see “Designing Protocols” on page 33.
Criterion Stain Free System | Editing Results 1. Image Tools Click to return to the Analysis Tool Box menu. FLIP You can flip your imaged gel horizontally or vertically. ROTATE You can rotate your gel image 90°, using the Left or Right buttons. To correct a slanted gel, click the Custom button and do the following: 1. Rotate the red arrows which appear over the gel to any degree between 0-360°, by clicking and dragging with your mouse. 2.
Analysis Toolbox 2. Lane Tools LANE FINDER You can choose how your lanes are detected, using the Automatic or Manual buttons. Either will delete previous lane detection. • Click the Automatic button if your gel image is fairly typical • Click the Manual button if you only want to detect a specific number of lanes, or if the automatic lane detection fails. You can then drag the handles on the corners of the red square, to resize the lane frame.
Criterion Stain Free System | Editing Results SINGLE LANE Add - You can add a single lane to your gel image. First click the Add button, then click within the lane frame where you want the new lane. The lanes will renumber. Delete - You can delete a single lane. First click the Delete button, then click either on the lane or its lane number. The lanes renumber. Move - You can move a single lane to a new position on your gel image. First click the Move button, then click on the lane you want to move.
Analysis Toolbox Delete - You can delete bands which are not relevant to your analysis. First click the Delete button, then click any bands you want to remove. Adjust - You can adjust the height of a band. First click the Adjust button. Two boundary lines appear around each band. Move your mouse over a boundary line until you see a double-headed arrow. Move the boundary line up or down, the center is recalculated, and the band appears there. 4.
Criterion Stain Free System | Editing Results 5. Annotation Tools ADD ANNOTATIONS Text - You can add text annotations to your gel images to draw attention to important details. First click on the Text button, then on an area you want to emphasize. A text box appears with a dotted-line border. Type your comment into the text box. Click and drag the box to change its position. Arrow - To add an arrow, first click on the Arrow button.
Analysis Toolbox Font - First click on the text box you want to change. Click on the pulldown Font menu to show all fonts installed in your system. Click on one of them to change the style of your text annotation. Size - First click on the text box you want to resize. Click on the up and down Size arrows to increase or decrease the size of your text. You can make text as small as 6 point and as large as 24 point.
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Criterion Stain FreeTM System 8 Generating Reports After viewing your Results Files, you can print the gel images with analysis, and include information about the protocol used and all other settings that were applied. The print report settings can be chosen initially when setting up your protocol (Step 5, Print Report), or after analyzing and viewing your Results Files. Report You simply click the Report icon to produce a Print Preview of your report.
Criterion Stain FreeTM System | Generating Reports Clicking the Print icon printer or .pdf file. allows printing of all of the above information to a The Report screen includes the following icons: Display Report Options Shows options of what to include in your report; simply uncheck a checkbox to remove an item. This dialog shows all information included as a default.
Report IMAGE INFO • • • • Acquisition Information Analysis Settings Image Information Notes INCLUDE ANALYSIS TABLE Lanes to show: • All lanes or user-defined • All lanes on one page, or one lane per page (add page break after each lane Show Lane Profile Print one Lane per Page Columns to Show: • Band Number • Band Label • Relative Front • Relative Quantity • Molecular Weight • Band Quantity Print Report You can select or add printers, and set the number of copies you want here. Print Report to .
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Criterion Stain FreeTM System 9 Exporting Results The most convenient way to keep complete information about your experiments is by producing Reports, which is discussed in Chapter 8. But you may want to export just your gel images or Analysis Table data for analysis in different programs, such as InfoQuest FP or Quantity One®. Or you may need exported files for presentation or publication. This chapter describes all the ways Image Lab can export your results.
Criterion Stain FreeTM System | Exporting Results See below for which file format to use for each type of export: • Raw Image - Use a .tif when you want all data to be retained. This option creates a larger file, but allows you to analyze the image in other software such as InfoQuest FP or Quantity One® • Displayed Image - You can choose between .bmp, .png, and .jpg formats, when exporting a displayed image. The gel will display with any lanes, bands, and annotations. • • Exporting as a .
Criterion Stain FreeTM System Appendix A: Troubleshooting Follow the recommendations below to troubleshoot problems with the Imager.
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Criterion Stain FreeTM System Glossary The following glossary defines words used in this Criterion Stain Free System manual. The equations used to calculate regression methods are also in this appendix, see “Regression Method Calculations” (page 75). 2-Mercaptoethanol (BME): A protein denaturant which breaks disulfide bonds. CCD: (Charge-Coupled Device) A light-sensitive silicon chip used as a photodetector in digital camera systems. Charge-to-Mass Ratio: The value measured by electrophoresis.
Criterion Stain FreeTM System | Glossary Native charge density: The inherent electrical charge of a protein without the addition of SDS. PAGE: Polyacrylamide gel electrophoresis; an electrophoretic method in which molecules migrate through a molecular lattice created by polymerized, cross-linked polyacrylamide. pl: Isoelectric point; the pH at which a protein molecule carries no net charge.
Regression Method Calculations Each regression method will calculate a standard curve. Some of the methods provide the formula for the standard curve. In this case, the molecular weight can be calculated by: x = relative front of the band of interest, y = molecular weight of the band of interest. Linear (semi-log): The linear equation is: y = a + bx, where a is the intercept and b is the slope of the line. Note: The linear equation is calculated on the log of the molecular weight values.
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Criterion Stain FreeTM System Index Numerics 2-Mercaptoethanol defined B 73 A Acquire Image Step 1. Activate Gel 35 Step 2. Image Gel 36 Analysis Table change orientation 51 copy table to the clipboard 52, 70 export to a file 52, 70 export to a spreadsheet 52, 70 Analysis Toolbox Annotation Tools 62 Auto Analysis 55 Band Tools 60 Image Tools 58 Lane Tools 59 Mol. Wt. Analysis Tools 61 Analyze Image Step 3. Detect Lanes and Bands 37 Step 4.
Criterion Stain Free Gels catalog numbers 15 comb configurations 15 description 4 distorted bands 22 formulations 15 reagents 24 running 16 setting up 16 specifications 16 storage 22 types available 15 Criterion Stain Free Imager description 4, 11 G D N Default Protocol running 3 discover.bio-rad.com ii discover.bio-rad.
Results Files editing 55 overview 43 viewing 43 viewing as a 3D projection 48 viewing as a Lane Profile 53 viewing as a Report 54 viewing as a Standard Curve 54 viewing as an Analysis Table 50 viewing Image Information 48 Results Files defined 25 S Safety iii Sample Tray cleaning 13 SDS-Page Buffers 19 charge variability 18 conditions 18 description of test 18 determining molecular weight running conditions 19 sample preparation 19 software installing drivers 7, 9 support.bio-rad.
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