Imaging Gels Stained with SYPRO^™ Ruby

Equipment:

• Perkin-Elmer ProXPRESS™ Proteomics Imaging System with ProFinder v.3.2.0.1

Method:

A. Flat field correction:

1. Place the gel adapter plate (the glass plate) onto the magnetic spacer and the Y ruler;
2. Lay the red acrylic sheet on top of the adapter plate;
3. Use top illumination mode as illumination method;
4. Choose the settings as follows:
1. Excitation filter: 480/30 nm
2. Emission filter: 530/30 nm
3. Resolution: 100 um
4. The area of the image:
   • Width=267, Height=210 with an Offset set at X=Y=0
5. Select “Flat field” option from the Acquire menu;
6. Change the flat field exposure time to achieve a value of ~30,000 pixel intensity across the center of the image;
7. Save the image of flat field correction with all the conditions:

(Example: x0y0Ex480Em530Exp3000ms100um)

B. Imaging proteins in gels:

1. Place a gel onto the glass plate;
2. Cover the gel with the wet gel cassette;
3. Lay the entire set onto the magnetic spacer and the Y ruler;
4. Note: for 1mm thick gels, use the magnetic spacer and the Y ruler with the white dots;
5. Set the X and Y offset based on the position of the gel;
6. Choose the settings as follows:
1. Excitation filter: 480/30 nm
2. Emission filter: 620/30 nm
3. Resolution: 100 um
4. The area of the image depends on the size and position of a gel
7. Use “Top” illumination mode as the illumination method;
8. Select “Image” option from the Acquire menu;
9. Change the exposure time to achieve a value of ~55,000 to 63,000 pixel intensity on two to three of the most intense spots on the gel;
10. Save the images according to the following procedure:
1. Create a folder with the investigator’s name;
2. For each service request from the same investigator, create a folder with the service request number, and use Save As to save images under this folder;
3. For requests for Image Analysis, create a folder under the folder with the same service request number called "non-linear";
4. Export the images into the non-linear folder as .tiff files.

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