Hi everyone!
I got a bit of an issue with my radioligand receptor binding. I've started with standard kinetic and saturation experiments to determine the incubation time and Kd. The thing is that in the kinetic experiment the association curve increases linearly and does not reach steady state even after 12h of incubation at 35deg.! The same thing happens with the saturation curve, that refuses to go hyperbolic. The non-specific binding (hot and cold are the same molecule) is about 20% of total binding at 60min. incubation in the association experiment so at least it competes with the hot ligand for the binding site. I tried to increase ligand concentration, buffer composition and pH in hopes that it will equilibrate faster, but no luck so far. Has anyone of you guys experienced this problem? I was thinking about running a homologous competition experiment, but I do not know whether the hot does not bind to several binding sites or there is no cooperativity.
There are a number of questions that come to my mind that need an answer first:
- How do you separate bound and free radioligand?
- What kind of ligand are you studying (small molecule, peptide, lipid, etc)?
- Are you studying binding to cells, membranes or solubilized receptors?
- What is the concentration range you are using in your saturation experiments? What is the expected Kd?
- Once the problem in the saturation experiments is pinpointed I assume the issue with the kinetic experiments is solved as well.
Two general comments that are most likely unrelated to your problem:
- I always prefer to incubate at lower temperatures (20-25°C) to avoid or minimize problems caused by protein damage at higher temperature.
- For the determination of nonspecific binding a cold ligand that is structurally unrelated to the radioligand should be used. Otherwise you might also detect 'false' specific binding of your radioligand to other targets than your protein of interest.
Thank you Karl-Norbert for you answer! As for your questions
- I use the filtration method for separation using the Brandel manifold and GF/C filters. I chose that method because it worked much better than centrifugation for my previous opioid binding studies. I was also afraid that centrifugation will not readily separate the bound from free and there is also a risk of disrupting the equilibrium.
- the ligand is a small, non-peptide drug-like molecule
- the source of the binding site is the mitochondrial brain fraction
- the concentrations I used so far ranged from 0.03nM-100nM. I don't know what the expected Kd might be as I'm investigating a new binding site
- I started with incubating the samples at RT, but the reaction didn't reach equilibrium, so I thought about going for 37 deg. Now I know that incubation temperature does not make any difference, so I switched to 25deg again.
- As for the cold ligand issue: I do not really know what the binding site is so the only choice is to use the structurally related ligand.
I think that in my next saturation experiment I'll increase the range of radioligand concentrations and see what happens.
One problem might be that for some reason your ligand accumulates slowly in the mitochondria. Maybe you should prepare mitochondrial membranes and see whether you find saturable binding there. This would eliminate ligand trapping if that were the problem.
I would not increase radioligand concentration further. If the affinity would be that low (Kd above about 30 nM) it is notoriously difficult, usually impossible, to measure reasonable binding.
Good luck!
Thanks for all your valuable suggestions! I'll try isolating and purifying mitochondrial membranes.
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