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Radioactive binding assay

by tahaha
Tags: assay, binding, radioactive
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tahaha
#1
Nov2-13, 04:27 AM
P: 5
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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mfb
#2
Nov2-13, 07:10 AM
Mentor
P: 11,890
Just a guess: radioisotopes are expensive, and can be problematic in high concentrations.
epenguin
#3
Nov3-13, 08:43 PM
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P: 1,985
Quote Quote by tahaha View Post
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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