Determination of ACE retardation coefficients (Rs). 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.