1. Plot of BOUND against FREE ligand
This is
TOTAL BINDING
= specific + non-specific
BOUND (moles/mg)
CONC FREE (M)

2. Calculation of Kd and Bmax
Cannot calculate from this plot
Need to isolate specific binding (the binding to receptors)
Carry out a second set of experiments this time in the presence of a high conc (1000 x higher) of another non-radiolabelled ligand
This will produce a plot of non-selective binding only

3. Plot showing non specific binding
TOTAL
NSB
BOUND (moles/mg)
CONC FREE (M)

4. Plot showing specific binding
TOTAL
NSB
SPECIFIC
BOUND (moles/mg)
CONC FREE (M)

5. Bmax & Kd from specific binding
BOUND (moles/mg)
4 x 10-15
2 x 10-15
5 x 10-9
1 x 10-8
1.5 x 10-8
2 x 10-8
CONC FREE (M)

6. Bmax & Kd from specific binding
BOUND (moles/mg)
4 x 10-15 Kd
2 x 10-15
5 x 10-9
1 x 10-8
1.5 x 10-8
2 x 10-8
CONC FREE (M)

7. Problems? OK for estimating Bmax
But rather inaccurate for estimation of Kd
Better if we had a linear plot
Therefore use Scatchard Plot
BOUND (x axis) v BOUND/FREE (y axis)
Bmax from intercept on x axis
Kd from gradient of the line

8. Scatchard plot
Gradient = 1 Kd
BOUND / FREE
Bmax
BOUND