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

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

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

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

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)

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)

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

Scatchard plot Gradient = 1 Kd BOUND / FREE Bmax BOUND