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Keys to Successful Densitometry

pixel values are not saturated, integrated density is directly proportional to concentration. Determining the concentration of an unknown sample is achieved by comparing the integrated density of that sample to a standard curve generated from the integrated densities and concentrations of known standards. For accurate determination of concentration values, users should take precautions so that pixels within bands of interest do not become saturated during acquisition and that the background is defined properly. After the image is acquired, determining the concentration of an unknown spot or band using Eagle Sight software version 3.0 begins by depressing the Concentration function key from the toolbar or Analyze menu (see figure 1 (30k),, GREEN). The software then prompts the user to set the background level. Defining an appropriate background level for concentration analysis entails the same steps as defining the background for integrated density analysis.

figure 2

Once a background level is set, the user can define standards either manually or automatically from the Concentration Calibration menu (figure 2 (27k),). In cases where accuracy is of the utmost importance, it may be necessary to manually detect standard bands in order to keep analysis areas identical throughout the procedure. Automatically defining standard bands is faster but can result in different sizes of analysis areas. After the standards have been defined, the standard curve can be plotted and viewed to determine how well the curve fits the data. If the default curve does not fit the standard data points well, the
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