I am trying to look for a protocol for measuring Kd value of receptor-ligand binding in HEk293 cells. Fortunately, cells endogenously express the membrane receptor of interest. I have alexa fluor labelled ligand. Last time I tried plating cells (5 x 10^4) in 96 well plate. Treated cells with alexa fluor labelled ligand for 2 h at 4 degree C. Removed the ligand, washed once with PBS, and there was absolutely no fluorescence. Should I be using some sort of a binding buffer to facilitate binding? Would poly-L-lysine coating of plate prior to plating cells would help? May be cells got washed off in washing. May be binding is too weak, can cells be cross linked with PFA at the end, before plate reading? Or, should I be doing everything with membrane factions and not whole cells?
Are you using fluorescence microscopy, flow cytometry, or a fluorescence plate reader to detect the fluorescence? From your description, it sounds like you were using a plate reader. This method may not be sensitive enough to detect the fluorophore in only 50,000 cells. (Of course, you should make sure the cells remained in the well, using a microscope.)
In any case, the method you are using would never allow you to measure the equilibrium dissociation constant of (Kd) of the receptor-ligand complex, because the measurement is not made under equilibrium conditions, due the the use of a washing step to remove unbound ligand.
I suggest you employ flow cytometry. It is a very sensitive method of measuring fluorescence because it is laser-based. I'm not sure it is really an equilibrium measurement (maybe if you don't remove unbound ligand?), but at the very least it should tell you whether the ligand is binding to the cells. In this case, you have to detach the cells from the surface. It would be a good idea to have a negative control: cells that do not express the receptor.
It might be possible to use confocal fluorescence microscopy (or a high-content screening instrument) to measure the Kd, by quantifying the amount of fluorescence associated with the cell membranes without washing out unbound ligand. The receptor concentration must be much lower than the Kd in order for this to be accurate. The cells would have to be grown on glass slides for microscopy.
Thanks for your response Adam B Shapiro . I do not have access to flow cytometry right now. But I can surely try confocal. yes, I had used a florescence plate reader for my previous trial. I was thinking of having a 'no-receptor expression' as my control. By treating cells with siRNA.
My first experiment was a trial run, just to verify that increasing amount of ligand gave me increasing levels of fluorescence. Unfortunately, I did not see that. Everything was back to background levels after washing. So, should I not be doing washing? But then, how do I distinguish between bound and unbound ligand? If I do not do washing, of course an increasing amount of ligand is going to give me increasing amount of fluorescence, since increase in ligand = increase in fluorophore.
For now, how do I confirm that ligand is indeed binding to the receptor?
With no washing step, when measuring the bulk fluorescence in the well of a plate, all you will measure is the total fluorophore concentration. There will be no way to discern the proportion of it that is bound, which will be a very small proportion in any case.
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