Image Analysis using Nonlinear Progenesis Software
Note: This protocol is for analysis of images scanned using the Perkin-Elmer ProXPRESS Profinder Proteomic Imaging system. The images must be scanned at a 100 micron resolution level and saved as .tiff files.
1. Start Progenesis Discovery software and select Automatic Analysis from the top menu
2. Give the experiment a name designating the PI name and the Service number (if any), with version and stage numbers
3. In the Analysis Wizard Dialog box, navigate to the .tiff images saved in the relevant folder and select all images that are to be analyzed using the software.
4. Adjust grayscale intensity of the images and set the Area of Interest (AOI) by drawing the green colored border for the areas of the gels where spots are to be detected. Repeat for each gel.
5. If Average gels are to be created from sets of replicate gels, group the gels accordingly. Any number of gels may comprise any number of Average groups. Name the Average group accordingly (Control, treated, Knockout, etc).
6. Set the Averaging parameters:
a. Set the Maximum number of gels in which spots may be absent. The default value is 0, which means that a spot must be present on all of the sub-gels before it is represented in the Averaged gels. The value set depends on how reproducible the spots on the gels appear.
b. Decide whether to Represent absent spots in calculations, this factor will effect how the average values are calculated.
c. You may select to Reject averaged spots which do not meet predefined tolerances for various spot measures (requires a priori knowledge)
7. You may elect to load a previously saved analysis protocol for experiments that are run in a similar fashion
8. Select a Reference gel for automatic matching. Typically, the best option is to let the software decide the gel with the most spots by default.
9. Choose Progenesis background as the method for Background subtraction (this method calculates a background level based on a surface model of the entire gel).
10. Select the Warping options: Warp to Reference gel and matching of detected spots to spots in the Reference gel. You may choose to perform combined warping and matching and also use Cross-gel detection, options that result in better matching and warping of spots across gels but which can take considerably more time.
11. Select to add unmatched spots to the reference gels to allow the reference gel to be inclusive of all the spots on all the gels in the experiment. Select to Synchronize spot numbers, giving the same spot number to all spots in a match series.
12. Normalize spot volumes using the Total Spot Volume method and multiply by 1,000,000 (Do Not select the option of using only spots that are present in all gels since this requires highly reproducible gels).
13. You may want to export to an XML file or a Database
14. You can set-up another experiment to run sequentially or start the automatic analysis at this point.