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Figure 5
Determination of ACE retardation coefficients (R’s).  To determine the retardation coefficients (Figure 2) we need to measure the mobility of the PG front, taken to be the distance from the top of each lane to the “midpoint” of the PG band. To determine the midpoint of the PG band, relative AP-staining intensity along a 2 mm wide vertical band in the middle of the lane is measured as a function of the distance from the top of each lane.  A typical plot of AP-staining intensity versus migration distance is shown in (A).  The staining intensity ranges from 0 (lightest) to 255 (darkest).   The midpoint of the band is taken to be the point, or line, that divides the area under the intensity curve into equal halves (shown as the dashed line), after subtracting the background intensity level (about 50 in this case).  In some cases, significant amounts of non-binding AP-positive degraded products were present (Figure 4, arrow), which register as a secondary peak on the intensity curve (B, top curve).  The degraded products are accounted for by subtracting from the original intensity values the intensity curve due to the degraded product alone (bottom curve) which yields a more accurate intensity curve (middle curve).  The midpoint of the corrected curve is then determined as described.

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