Bio-Rad PDQuest 2-D Analysis Software User Manual
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Section 3
System Calibration
The interaction between the software, the computer, the camera and the spot cutter requires an
initial calibration to bring these parts into alignment. The software drives the calibration process.
The user loads templates and confirms to the software that a step is ready. Then the software
completes the step, and at the end the system is calibrated and ready for use. The calibration
process should be run at initial setup, and then re-calibrated if the instrument or camera is
moved, or if there is a change in the accuracy over time. Normal use should maintain system
accuracy for over 6 months to 1 year. To calibrate the x, y coordinates a 28 x 26 cm Coomassie
stained PVDF membrane is needed.
3.1
Switch to White Light Imaging
In preparation for the x,y calibration steps, set the instrument to the White Light mode.
•
Switch the light toggle switch to the "lights on" position. Test the lights by pushing the red
"Lights on" button on the lower front right side of the spot cutter. If the platform lights do
not come on, switch the toggle switch to the other position.
•
Set the aperture to f-stop 11-16.
•
Take the cutting mat, and any other materials, off 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".
3.2
Prepare Coomassie Stained PVDF Membrane
1. Your spot cutter shipment included 1 package of precut PVDF membrane, 25 x 28 cm,
and 1 L of Coomassie stain solution. Use these materials for the following procedure. If
you do not have these materials, you can order them using Bio-Rad catalog numbers:
•
162-0255, Immun-Blot PVDF membrane, 25 x 28 cm
•
161-0436, Coomassie Brilliant Blue R-250 Stain solution, 1 L
Alternatively, you can use any PVDF membrane cut to the correct size for the spot cutter
platform, and stain with a Coomassie R-250 solution.
2. Obtain a tray with dimensions of at least 26 x 29 cm. Pour enough Coomassie R-250
stain solution into the tray to completely cover as many PVDF membrane pieces as you
are preparing.
3. Wet the PVDF directly in the Coomassie R-250 solution by putting the PVDF in by its edge
first and sliding the membrane into the solution.
4. Shake or rock the tray for 10-20 minutes. The membrane should be a uniform deep blue
color.
5. Remove the Coomassie solution and wash with water until excess Coomassie stain is
removed. (Do not use destain solution).