Method 2 - Comparing two 2D gels (DIGE-like method)

With this method, two gel images are displayed superimposed in different colors (red and green). You then manually warp the green image to maximize the overlap. Since overlapping spots appear yellow, this procedure amounts to maximizing the amount of yellow on the screen. At this point, any spots that are increased appear green, and any spots that are decreased appear red. You then go to spot densitometry and measure the density of the spots that have changed.

This process is similar to differential gel electrophoresis (DIGE), except that the control and experimental samples do not need to be on the same gel. For example, we use this method in our laboratory to analyze 2D gels stained with Sypro ruby. See http://brneurosci.org/sypro.html for more details. If the two samples are in the same gel, the warping step can obviously be omitted, which makes analysis much easier.

The advantage of this method is that it's much easier and faster to go back and measure only the spots that are different. Instead of having to measure thousands of spots per gel, you only need to measure a few dozen. This also eliminates the statistical problems that arise when you compare such large numbers of independent spots. If you measured 2,000 spots, you would need to use MANOVA to ensure that you don't get spuriously significant results.

To make sure you catch all the spots that have changed, click on Capture image and use Image Math to highlight all areas where red is a certain percentage higher than green, or vice versa. Even easier is to select "Highlight differences", which does this automatically.

1. Acquire two images (control + experimental or control 1 + control 2) under identical lighting conditions.
2. Apply flat-frame correction if desired (see Sec. 8.3).
3. Open the dialog box ``Process ... Manual Image Registration''.
4. Specify the control (``reference'') image as Image no. 1 and the second control or the experimental (``unknown'') as Image no. 2. (Comparing two controls is a good way of testing for variability).
5. Accept the defaults (Grid visible, Make ref image green, Make unk image red) and click Accept.
6. A blue grid will appear, similar to the blue grid that is used to warp an image. Dragging on the vertices of the grid will warp the green image, leaving the red image untouched. Alternatively, click on a vertex and press the arrow keys to move the grid point by a specific distance. The distance it moves can be changed in the Cursor Movement clickbox.
7. At any point, clicking on ``Capture image'' will create a new color image showing the current overlap.
8. You can also save the current warping map into a file, and read it back later. This can be useful for continuing an analysis later or refining your gel warping. Click "Apply warp to current image" to warp the currently-selected image. Warning: do not click this button while you're in the middle of an analysis. This would cause the image to be warped twice.
9. If the blue lines are too close together or too far apart, this can be changed in the Grid Size clickbox.
10. When the two gels are sufficiently overlapping, click on ``Capture image'' to create a color image of the overlapping images.
11. When finished, press the Esc key or click the Cancel button on the left of the main Imal window. This will turn off the pseudo red and green, and you will have the original reference image and a new warped ``unknown'' image.
12. Make a note of the spots that still appear red or green in the captured image, or use ``Highlight Image Differences'' to select them automatically.

When printing a color image of overlapped gels, better results are sometimes obtained if the image is color-inverted (Color ... Invert colors). Some printers have difficulty with solid black areas.