I would like to quantify the fraction of occupied receptors of a growth factor receptor at various concentrations of ligand and correlate it to a biological process. How do you go about this?
My (naive) plan was to make a fluorescent ligand using an antibody-conjugation kit (I still have a lightning link kit lying around). Then I would lift the cells, add my ligand to the cell suspension, wash and measure fluorescence on a flow cytometer. I'm kinda worried that I'd get bad results because of receptor internalization, but I figured that might be minimal if I kept the cells at 4 deg. after I add the ligand.
Also: does it really matter if the fluorescent conjugate is big? Easiest for me would be PE (which is 30kd in itself) because that is what I have in house, but I could always order a smaller fluorophore. Of course I would do a functional assay to determine if my ligand was still functional after conjugation.
So does anyone have tips on this? How is it usually done (except for radioactive ligands, that I can't do here)? Do people do the inverse and look at receptor internalization?
Thanks in advance for helping out, useful articles and protocols will be cited if this ever sees publication.
If I am correct, you have two concerns/questions:
1. Change of ligand activity upon conjugation: labeling can influence ligand activity. If you use PE, I would be certain that it does. PE is much larger than most soluble ligands of RTKs I know of. I would therefore use a smaller ligand (Alexa dyes). And even if you check that the conjugated ligand activates the receptor as the unlabeled one (using e.g. western blotting), you can't be sure that its affinity is the same as that of the natural ligand. Therefore, ligand displacement (i.e. competition of the labeled ligand with the unlabeled) should be done to compare the affinities.
2. Internalization: I think it is not a significant problem if you keep the cells at 4 degrees throughout the labeling and measurement procedure. Probably you can't adjust the temperature of your flow cytometer to 4 degrees, but 1-2 minutes of measurement will not result in huge internalization. You shouldn't use a FacsArray in which all the samples are placed in the device, so many of them will be measured after having spent 5-10 minutes in the device.
I would also consider the effect of ligand depletion.
Thanks Peter,
You're probably right about the PE, I guess I shouldn't even bother and go straight to an Alexa dye. Good to know that you think the flow cytometry will work in principle, I think I'll just go ahead and try this. We don't have a FACS array or similar, so I'll manually feed the machine anyway.
As for ligand displacement, that would be nice, but I can't find any competitive inhibitors for my target (PDGF-R) and I wouldn't begin to know how to make one.
BTW, can you also measure receptor occupancy by looking at receptor internalization (I guess by doing a similar flow cytometry experiment but then using an antibody against your receptor and looking at a decrease)? Will you ever see complete absence of the receptor in that case?
How does ligand depletion work? You just measure the reduction in available ligand with an ELISA or something?
Thanks for helping out,
Regards,
Hendrik
Hi Hendrik,
Regarding competitive binding experiments you should read the relevant chapter from the attached PDF.
There you can see that you basically compare the binding of your unlabeled ligand to that of the labeled one.
I don't think receptor occupancy can be measured by receptor internalization. Receptor internalization is never 100%, and receptors are also synthesized and exocytosed, so what you would need to analyze is a very complex system.
You could measure ligand depletion with ELISA or similar solution-based techniques. But most of these are not as accurate and quantitative as flow cytometry. I think you could simply determine the number of receptors on your cells (e.q. with Qifikit). You know how many cells you have in the suspension. You multiply the number of receptors/cell with the number of cells in the solution and then further multiply with the fractional occupancy. This will give you the number of ligands bound to your cells. If this is less than 10% of the total number of ligands you added, then ligand depletion isn't significant.
BTW you can read a lot about these concepts on the following site:
http://www.graphpad.com/guides/prism/6/curve-fitting/
Regards,
Peter
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