Why mix radioactively labeled ligand with normal ligand in a binding assay?

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The discussion centers on the methodology of a radioactive binding assay, which involves mixing a radioactively labeled ligand with a normal ligand before introducing it to a receptor solution. The purpose of this mixing is questioned, particularly why a solution of only radioactively labeled ligand cannot be used instead. One hypothesis suggests that radioisotopes are costly and problematic at high concentrations. The conversation also touches on the concentration of radioactively labeled ligands available commercially, noting that they often contain a small percentage of the radioactive component. Concerns are raised about the potential for non-specific binding if the concentration of the ligand is too low, especially in the context of irreversible versus reversible binding scenarios. The importance of matching ligand concentrations with binding site concentrations or affinity constants is emphasized as a critical factor in the assay's effectiveness.
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Here's a radioactive binding assay:

1. Mix radioactively labeled ligand of known concentration with normal ligand (assuming radioactivity does not affect the ability to bind).
2. Add the mixture of labeled and unlabeled ligand into the solution of receptor.
3. Separate bound ligand from unbound (by centrifugation).
4. Measure radioactivity of the receptor (which should be ligand bound). By comparing with the known concentration of labeled ligand, the fractional saturation can be calculated and a binding isotherm can be plot.

I don't understand why they have to mix the labeled ligand with normal ligand in the first step. Why can't they just use a solution of radioactively labeled ligand? You still get the portion of ligand bound by comparing with the known concentration in the beginning?
 
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Just a guess: radioisotopes are expensive, and can be problematic in high concentrations.
 
tahaha said:
Here's a radioactive binding assay:

1. Mix radioactively labeled ligand of known concentration with normal ligand (assuming radioactivity does not affect the ability to bind).
2. Add the mixture of labeled and unlabeled ligand into the solution of receptor.
3. Separate bound ligand from unbound (by centrifugation).
4. Measure radioactivity of the receptor (which should be ligand bound). By comparing with the known concentration of labeled ligand, the fractional saturation can be calculated and a binding isotherm can be plot.

I don't understand why they have to mix the labeled ligand with normal ligand in the first step. Why can't they just use a solution of ? You still get the portion of ligand bound by comparing with the known concentration in the beginning?

The "radioactively labeled ligand" already is such a mixture I think. A quick look in a Sigma co. Catalog showed me some typical biochemical tritiated products were being sold as 10-60 Curie/mmol (unbelievable they still use those execrable units). According to my fallible late night calculation that makes about 1% of the products tritiated when new but somebody check.

Still why not use that without further dilution? I do not know what the concentration of binding sites is. It sounds like you are talking about irreversible binding. For measurement something of order of 1,000 Bq are I think are OK. Again according to my fallible calcs that is 10-14 moles or 10-11M if done in 1 ml. I wonder if such a low number of moles wouldn't be bound unspecifically to any old protein around?

Anyway for irreversible binding you want amounts of ligand comparable with the amounts of bidding site; for reversible binding you want concentrations comparable with the affinity constant. I think the answer is probably around such considerations - if you are talking about a particular system about which there is some information you could see the numbers and whether the above makes any sense.
 

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