Chem 8157

7) Our Kd value of 7.62 nM was calculated and is in comparison to the Kd values obtained for the paper of 16nM and 12 nM (calculated for two different solvents). The %error of our value with the paper's values are 52.5% and 36.7%. Overall we felt are calculations are solid but with sources of error: we did not measure any of the mobilities present at 1nM [AT], we may have measured the points and normal band with a different method than Lee et. al., and our measurements are limited by the resolution of the photograph.

8) To determine the minimum Kd value that our method could reasonably detect, we used the standard error in the slope of our Scatchard Plot. We took the standard error (.009) multiplied it by the square root of the number of observations (8) to get the standard deviation (.0025). We took three times the standard deviation (.075) and subtracted it from our slope value (-.1313), which gave us -.2063. This represents the lowest possible slope that we can accurately measure. Since the slope=1/(-Kd), the lowest possible Kd we can measure using this method is 4.8 nM. We used a different method to determine the highest possible Kd obtainable. From the gel given in the paper we measured the furthest possible migration that we thought we could reasonably measure at a concentration of 1000 nM. From this measurement we determined the R factor for this Kd and concentration (.1319). For our point we assumed that at a concentration of 1 nM the R factor is going to be really low, since the amount of hepatin bound will be so small it will essentially be like free hepatin moving through the matrix, and will therefore have an R factor of close to zero. To get an accurate Scatchard Plot we needed to assume that our R factor would not be smaller than the R/[Free Protein] factor. This meant estimating an R factor of .00014, which is very close to zero. We created another Scatchard Plot for these two points and from the slope of that graph determined the maximum Kd, which we found to be approximately 16210 nM.