competitive receptor binding assay with opposite results what is expected

Laszlo G. Boros lboros at magnus.acs.ohio-state.edu
Thu Dec 21 09:15:44 EST 1995


We are trying to establish a competitive binding receptor assay for amylin (pancreatic islet
polypeptide) on pancreatic carcinoma cell lines in our laboratory.  We are using 5000 uCi
labeled amylin in  each experiment and we add 10-5, 10-6, 10-7, 10-8, 10-9, 10-10, 10-11,
10-12 and 10-13 uM cold, unlabeled peptide as competitors, separately.
  
The results are the opposite what we expect from the assay.  The highest concentration of 
cold peptide gives us the highest hot binding, decreasing concentrations of the cold
peptide results in decreasing hot peptide binding in the assay.  This is the opposite what
we have seen with other peptides where  high cc of cold peptide resulted low hot binding
because of competition for the binding sites.  We have contemplated the idea that amylin
might have an internal receptor and competition is occurs not  at the cell surface where 
the unbound hot peptide gets washed off easily after the experiment, or amylin binding is 
ot specific to receptors but the osmotic pressure will direct the hot peptide to the cell
surface or into the cells.  If anyone ever saw the same binding pattern in their experiment,
comments are welcomed and very much appreciated.  Thanks, Laszlo G. Boros, MD  The 
Ohio State University Dept. of Surgery.







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