Image Lab™ Software How to Obtain Stain-Free Gel and Blot Images Instructions
Table of Contents Obtaining Stain-Free Gel images ...............................................................................1–5 Obtaining Stain-Free Blot Images ..............................................................................
If the Gel Doc™ XR+ or ChemiDoc™ XRS+ systems are used to conduct quantitation with Bio-Rad’s stain-free gel technology, Bio-Rad recommends performing the flat field calibration with the orange fluorescence reference plate (cat. #170-8008) after installing Image Lab™ software, version 5.1 or higher. To perform the calibration using the orange fluorescence reference plate, choose Edit > Instrument Setup in the Image Lab software and select Reset next to the flat field in the dialog box.
4. In the Application pane, click Select and then Protein Gels > Stain Free Gel. Now select the gel activation, depending on the application. a. 1 min activation provides sufficient UV activation for gels used in western blots. b. 2.5 min activation provides a good balance between time required for UV activation and signal intensity. c. 5 min activation provides maximum signal intensity. 5. In the Imaging Area pane, select from the list of Bio-Rad gels or enter the image area dimensions manually.
6. In the Image Exposure pane, select from one of the following options: a. Auto Exposure — this setting estimates an optimal exposure time and ensures the best use of the dynamic range. i. ii. Intense Bands — optimizes exposure for all bands. Faint Bands — a longer exposure time is used to make faint bands more visible, but more prominent bands might be overexposed. b. Manual Exposure — use this setting to manually override automated imaging.
10. To start the stain-free activation and image acquisition, select Run Protocol. 11. The Gel Image Preview pane will now display the stain-free gel undergoing activation in real time. 12. Once activation is complete, an image of the stain-free gel is captured, based on the image exposure time selected.
13. The gel image can now be exported in a variety of image formats (TIFF, JPEG, bitmap, or PNG).
Obtaining Stain-Free Blot Images Instructions 1. Once the blotting step is complete, remove the blot and place it directly on a stain-free enabled Bio-Rad UV transilluminator plate (ChemiDoc MP, ChemiDoc XRS+, Gel Doc XR+) or stain-free tray (Gel Doc EZ). Note: No fixation or rinsing steps are required. Membranes are more transparent to UV illumination when wet. For optimal imaging, keep membranes wet with transfer buffer or water.
5. In the Imaging Area pane, select from the list of Bio-Rad gels or enter the image area dimensions manually. This option is not necessary when using the Gel Doc EZ, as the entire area is imaged automatically. 6. In the Image Exposure pane, select from one of the following options: a. Auto Exposure — this setting estimates an optimal exposure time and ensures the best use of the dynamic range. i. ii. Intense Bands — optimizes exposure for all bands.
7. In the Display Options pane, keep the default settings. If using the Gel Doc EZ, proceed to step 10. 8. Select Position Gel. The acquisition pane will now display a live view of the blot. 9. Make sure the blot is correctly positioned in the viewing area. Adjust the zoom if necessary and/or manually align the blot. 10. To start the stain-free blot and image acquisition, select Run Protocol. 11. The Blot Image Preview pane will now display the stain-free blot undergoing image acquisition in real time.
12. Once image acquisition is complete, an image of the stain-free blot is captured, based on the image exposure time selected. 13. The stain-free blot image can now be exported in a variety of image formats (TIFF, JPEG, bitmap, or PNG).
Notes and Tips If desired, stain the gel with any TGX-compatible stains after stain-free imaging. Certain stains eliminate detection capability if used prior to imaging. Wet the UV transilluminator with 2–3 ml of water prior to gel placement in order to avoid tearing. If uneven stain-free development is observed, briefly rinse the gel with water after electrophoresis. However, prolonged rinsing may affect the intensity of the stain-free signal.
Image Lab Software for Obtaining Stain-Free Gel and Blot Images 11
Bio-Rad Laboratories, Inc. Web site www.bio-rad.
