separating bound vs free ligand question

[chef]

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After I solubilize and purify insulin receptor from human placentae using sepharose-insulin column chromatography, I'd want to confirm the bioactivity and affinity of the solubilized, purified receptor. I could do a binding affinity experiment using 125I labelled insulin in the presence or absence of unlabelled insulin.

My question is, now that the insulin receptor is solubilized, how do I separate the free vs bound labelled insulin before measuring the radioactivity??

 

[Nuel]

Try doing a filtration or some affinity chromatography. If your receptor has high affinity for a chemical on your chromatogram, then you try it. The only thing to watch out for is that your bound ligand doesn't get washed down the chromatogram. Only free ligand must be excluded. Or you could try doing microdialysis to specifically separate the receptor-ligand complex from the free ligand.