How can I estimate Kd value or any parameter showing the affinity of protein-protein interactions, If I have measurements(band intensities from SDS PAGE) only in linear range? Any methods, other than using saturation binding curves with Prism GraphPad?
Hi there,
If your binding curve (bound ligand=f(total ligand)) is linear and if total ligand is far more than total binding sites then you may state than Kd is far over the upper concentration of total ligand. If you also know the stoechiometry of interaction and the concentration of protein then you might make a calculation :
the binding equation is : [bound ligand]=[total binding sites]*[total ligand]/(Kd+[total ligand]) so if Kd>>[total ligand], equation gives
[bound ligand]=[total binding sites]*[total ligand]/Kd so the slope of the curve is actually [total binding sites]/Kd. As [total binding sites] = number of sites per protein* [total Protein] you can estimate Kd.
The receptor-ligand binding isotherm equation for a 1:1 stoichiometric interaction is fraction of receptor bound [RL]/[R] = [L]free/(Kd + [L]free)
where [R] is the receptor concentration, [L]free is the free ligand concentration, and Kd is the equilibrium dissociation constant. Both R and L are proteins in this case. The concentration of one protein is held constant (let's say it is R) and the other one (L) is varied. You measure the concentration of the complex ([RL]) as [L]total increases. Some of L binds to R, so [L]free < [L]total. If [R] [L]free. Nevertheless, it would be advisable to repeat the experiment with a higher range of [L] to get some amount of saturation, since it is difficult to be sure that Kd >> [L]free.
The classic Prisma MM equation assumes the total ligand concentration to be well above the concentration of receptor ([L]
You really should demonstrate saturation. Otherwise it's hard to be certain that you're observing specific binding. Maybe you can use a lower concentration of the capture/receptor protein to facilitate a higher ratio.
- Badges
- Science method