Imaging Analysis

The evaluation of the spot patterns is the most demanding part of the 2-D electrophoresis technique, and the images would be analyzed comparatively and automatically. 2-D gel patterns are compared to detect qualitative and/or quantitative protein expression changes between individual samples or different populations. Imaging analysis of 2-D gels can provide various types of information for the detection of novel, missing, or modified proteins; quantification of protein spots; determine of pI and Mr values of protein spots; enzyme digest and mass spectrometry detection.

For the evaluation and comparison of the complex 2-D patterns, the gel images have to be converted into digital data with a scanner or camera for further analysis with computer software. In imaging analysis, usually a pixel format of 100 mm is applied for scanning, higher pixel resolution does not improve imaging analysis and the image files will become too big to be processed in a reasonable time and take up a lot of space on the hard disk.

The scanner must be calibrated using a gray step tablet. The measured dimensionless intensity is converted into OD values or absorbance units. For accurate picking of spots according to the data of imaging analysis, the x/y positions need to be absolutely correct in the mm tolerance range. Usually the x/y data have to be calibrated for each scanner with the help of a grid. These calibration data have to be imported into the spot picker computer for each scanner used.

In imaging analysis of 2-D patterns, a background subtraction function can easily lead to errors. After many years of experience with spot volume quantification, it has been found that by defining the spot boundary at 75% of the peak maximum and calculating the spot volume for only the values above the boundary, the relative spot volume quantification comes very close to the real situation and ensures a very high reproducibility.

In normalization of the variation in protein loading and

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