Analysis of simple binding assay

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dimps
dimps's picture
Analysis of simple binding assay

Dear all,
Please could someone help, I have conducted a very simple radioligand binding assay using tritinated nms and CHO cells. I used one concentration of ligand and also use a nonradioactive ligand of atropine to assess specific and nonspecific binding. My results are as expected but need some help with analysis. I have told to work out Bmax, kd etc but i thought that you could work out Bmax only when you use varying concentrations of agonist. In my assay this was constant. Can anyone please please remind me of the basics again as all the info on the net refers to bmax and varying concentrations of agonist! thank u ever soo much!

R Bishop
R Bishop's picture
dimps,

dimps,

Can you give some more information as to what you varied in your assay? was it time? You cant calculate Bmax or kd without varying concentration over time, so Im a little confused as to what you are asking.

Rus

smbgpd
smbgpd's picture
Start again and plan the

Start again and plan the experiment properly so you can answer the questions you are asking.

Bluejay
Bluejay's picture
My understanding is that you

My understanding is that you can only get a Ki from your experiment.  A Kd and Bmax calculation would require diluting off your hot agonist ligand with cold agonist ligand, getting a displacement curve, and then having a program such as GraphPad calculate the Kd.  Traditionally, people perform a Scatchard plot, looking at binding with variable amounts of hot ligand, plotting Bound/Free (yaxis) vs. Bound (xaxis).  Ki can be calculated using Cheng-Prushof equation of Ki = IC50/ (1 + (L/KD))   Cheng Y, Prusoff WH (December 1973). "Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction". Biochem Pharmacol 22 (23): 3099–108. PMID 4202581