For Fluorescence-Based Competitive Binding Assays

This web page provides a mathematical equation
that is particularly developed to convert *IC _{50}* values observed in
fluorescence-base competitive binding assays into

## The inputs: How the FP-based binding assay was performed |

We assume a typical case of fluorescence-based competitive binding assay.
Let ** P** denote for the protein
molecule,

Let [*P*], [*L*], [*I*], [*PL*],
and [*PI*] denote for the concentrations of these five species,
respectively. Let [*P*]_{T}, [*L*]_{T}, and [*I*]_{T}
denote for the total concentration of the protein, the ligand, and the
inhibitor, respectively. Let *K _{d}* denote for the dissociation
constant of the

**Please make sure that your binding assay meets the
following basic assumptions before you apply the equation below.**

(1) We assume that ** I** inhibits
the binding of

(2) We assume that ** L** and

(3) We assume that a positive control, which
is a mixture of fluorescence-labeled ligand ([*L*]_{T}) with the
protein ([*P*]_{T}) defining the maximal FP signal (*FP _{max}*),
and a negative control, which contains only the fluorescence-labeled ligand ([

The equation below will be used to compute the *K _{i}*

The above equation shows that *K _{i}* can be expressed as a
function of the concentration of the free inhibitor at 50% inhibition, [

[1] Renxiao Wang, Zaneta Nikolovska-Coleska, Xueliang Fang and Shaomeng Wang, "From
*IC _{50}* to

[2] Zaneta Nikolovska-Coleska, Renxiao Wang, Xueliang
Fang, Hongguang Pan, York Tomita, Peng Li, Peter P. Roller, Krzysztof Krajewski,
Naoyuki Saito,
Jeanne Stuckey and Shaomeng Wang, "Development and
Optimization of a Binding Assay for the XIAP BIR3 Domain Using Fluorescence
Polarization", *Analytical Biochemistry*, **2004**, *(accepted for
publication).*

* This page is constructed and maintained by Dr. Xueliang Fang and Dr. Renxiao
Wang. Latest update on 04/12/2004.

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times.