In article <lboros.16.30D96C0F at magnus.acs.ohio-state.edu>,
lboros at magnus.acs.ohio-state.edu (Laszlo G. Boros) wrote:
> 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
> cold peptide gives us the highest hot binding, decreasing concentrations
of the cold
> peptide results in decreasing hot peptide binding in the assay.
I am sorry, but the notation was not clear...with 10-5 you mean 10 to the
*minus* 5 (i.e. 1/100000) uM as the highest concentration? This is an
awfully low concentration. Not to speak about 10-13. On the other hand, if
you mean 10 to the fifth uM as the lowest, it's already 100 mM...so I
think the first guess was true. If so, how much excess (as molar ratio) is
it of cold with respect to labelled peptide (5000 uCi does not say
anything about the actual molar concentration)? If you are adding 1/100000
uM cold, it is possible that this low concentration does not compete out
your hot peptide, but instead allows you to see a cooperative effect at
the receptor level (I mean, in case of multiple binding sites, saturation
of the first site increases the affinity of the second site and so on...).
This, especially if you are using a subsaturating amount of hot one. Do
you know the Kd of the receptor for its substrate? If so, is the
concentration of the hot peptide lower or higher than that value? I would
suggest you to repeat the experiment by: 1) using a concentration of hot
peptide almost equal to the Kd of the receptor for the substrate; 2) using
cold peptide at 0.01, 0.05, 0.5, 5 Kd concentrations. In this way you
should see a competition. Non-linear regression analysis of your data
fitting a simple or multiple site binding equation should give you a clue
about the existence of only one or multiple sites.
Hope it helps. You can post the same question also in
bionet.molbio.proteins.7tms_r (which stands for 7 Trans-Membrane domainS
Receptors). There you'll find receptor experts.
Institute of Veterinary Biochemistry
University of Zuerich-Irchel (CH).