How does fluorescence polarization assay work?

Basic principle of fluorescence polarization. A fluorophore is excited with light that is linearly polarized by passing through an excitation polarizing filter; the polarized fluorescence is measured through an emission polarizer either parallel or perpendicular to the exciting light’s plane of polarization.

Why Fluorescence Polarization Immunoassay is important?

Fluorescence polarization immunoassay (FPA) is a homogeneous immunoassay useful for rapid and accurate detection of antibody or antigen. Because it is a primary antigen-antibody interaction, the rate of reaction is very rapid and usually a result may be obtained in minutes.

How do you measure fluorescence anisotropy?

It can be measured by moving the excitation polarizer to the horizontal orientation and comparing the intensities when the emission polarizer is vertically and horizontally polarized respectively. G is emission wavelength dependent. Note G in literature is defined as the inverse shown.

What is mP in fluorescence polarization assay?

Fluorescence polarization G factor Polarization, expressed in units of milli P, or mP, is calculated from the measurements of perpendicular (Iperp) and parallel (Ipara) fluorescence intensity values detected relative to the direction of the polarized excitation light (see the formula below).

What is an FP assay?

Fluorescence polarization (FP) technology is based on the measurement of molecule rotation, and has been widely used to study molecular interactions in solution. This method can be used to measure binding and dissociation between two molecules if one of the binding molecules is relatively small and fluorescent.

What does fluorescence polarization measure?

Fluorescence polarization (FP) measures protein binding based on change in polarization of emitted light upon excitation of a fluorescent molecule with plane-polarized light.

How does fluorescence immunoassay work?

A modern fluorescent based immunoassay uses as the detection reagent a fluorescent compound which absorbs light or energy (excitation energy) at a specific wavelength and then emits light or energy at a different wavelength.

Is fluorescence isotropic?

Fluorescent samples typically emit isotropically in all directions. Large lenses and other optical components are needed to capture a significant fraction of the emission, and complex confocal microscopes are required for high resolution focal-plane imaging.

How do you calculate concentration from fluorescence intensity?

em(λ)dλ = Q.

  1. Dividing the fluorescence intensity of the target label Ftar (to be determined) by fluores- cence intensity of the reference label Fref (of known concentration) located in the same volume.
  2. V, we obtain: Ftar.
  3. Fref. = A.
  4. [Ltar] [Lref ]
  5. ,A = Etar.

What is drug and FP?

Fluorescence polarization assays (FP assays) are used in a variety of ways. From uncovering molecules in solution, to monitoring drug levels in clinical settings, and are enabling in drug discovery. Moreover, scientists use FP to study small molecule-protein, antigen-antibody, and hormone-receptor binding.

What is HTRF assay?

HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS).

What is FP protein?

Fluorescent proteins (FPs) offer scientists a simple yet powerful way to tag cellular proteins and investigate protein localization, interaction, and expression. However, one caveat of FP-protein fusions (FP-chimeras) is that they undergo normal protein turnover.

How is fluorescence used in protein binding assays?

This property of fluorescence can be used to measure the interaction of a small labeled ligand with a larger protein and provides a basis for direct and competition binding assays.

How are FP assays used in protein screening?

FP assays are readily adaptable to a high-throughput format, have been used successfully in screens directed against a wide range of targets, and are particularly valuable in screening for inhibitors of protein-protein and protein-nucleic acid interactions when a small binding epitope can be identified for one of the partners.

What is the principle of fluorescence polarization ( FP )?

The technique of fluorescence polarization (FP) is based on the observation that when a fluorescently labeled molecule is excited by polarized light, it emits light with a degree of polarization that is inversely proportional to the rate of molecular rotation.

Can a fluorescent assay be used for kinetic constants?

There are many techniques that dig deeper beneath the surface than just affinity, including fluorescence techniques, which are highly simplistic and low-cost methods for obtaining affinities and/or kinetic constants. However, there are a number of drawbacks when fluorescent techniques are the sole assays employed for obtaining binding information.