Assay Readouts Lecture 4

Question 1

Fluorescence Polarization Application

Answer 1

Fluorescence polarization enables the measurement of changes in polarized fluorescence emission between small free/unbound labeled ligands that tumble quickly in solution and labeled ligands bound to large receptors, which tumble more slowly.

Low mP signal - Small
High mP signal - large

Question 2

Dependence of Fluorescence Polarization on Molecular Mobility

Answer 2

1. Fluorescence polarization is inversely related to the speed of molecular rotation of that complex.
2. Fluorescence polarization increases as molecular weight increases
3. Fluorescence polarization increases as solvent viscosity increases
4. Fluorescence polarization decreases as the excited state lifetime of the dye (τ) increases.

Question 3

Readers for Fluorescence Polarization

Answer 3

Two Polarizers - Excitation polarizer and Emission polarizer

Experimentally, the degree of polarization is determined from measurements of fluorescence intensities parallel and perpendicular with respect to the plane of linearly polarized excitation light and is expressed in terms of fluorescence polarization (P)

Question 4

Advantages of Fluorescence Polarization

Answer 4

1. Automated
2. Rapid
3. Homogenous assays
4. Detection Limit in sub-nanomolar range
5. Reproducibility
6. Stable
7. Less susceptible to pH changes

Question 5

Disadvantages of Fluorescence Polarization

Answer 5

1. Nonspecific binding

2. Background fluorescence

Question 6

Applications of Fluorescence Polarization

Answer 6

1. Molecular orientation and mobility of:
   a. Receptor/ligand
   b. Protein/peptide interactions
   c. DNA/Protein interactions
   d. Tyrosine kinase assays
   e. Competitive Immunoassays
   f. Membrane fluidity and muscle contraction

Question 7

Fluorophores for Fluorescence Polarization assay

Answer 7

1. Fluorescein
2. Cy3
3. BODIPY

Question 8

Competitive Kinase Fluorescence Polarization assay

Answer 8

Unbound ligand rotates rapidly and light becomes depolarized.

Acceptor comes and binds to the ligand. Bound ligand rotates slowly and light remains polarized.

Acceptor and competitor are in solution. Competitor binds to the acceptor and the unbound ligand remains led to low polarization.

Question 9

Fluorescence Resonance Energy Transfer (FRET)

Answer 9

FRET allows detection of molecule-molecule interaction in nm range with light microscopy.

Transfer of energy without emission of a photon from a donor molecule to an acceptor molecule in close proximity.

FRET is a technique for measuring interactions between two proteins in vivo or detect the assembly, dissociation or conformational rearrangement of protein and nucleic acid complexes

Question 10

Primary Conditions of FRET

Answer 10

1. A distance-dependent interaction between the electronically excited states of two dye molecules. Donor and acceptor molecules must be in close proximity (typically 10–100 Å)
2. The emission peak of the donor must overlap with the excitation peak of the acceptor.
3. Minimal direct excitation of acceptor at the excitation maxima of the donor

Question 11

Donor-Acceptor Pairs for FRET

Answer 11

1. Fluorescein - Tetramethylrhodamine
2. IAEDANS - Fluorescein
3. EDANS - DABCYL
4. Fluorescein - QSY-7 dye
5. BFP - GFP
6. CFP - YFP

Question 12

R0 values

Answer 12

R0 values in angstroms (Å) represent the distance at which fluorescence resonance energy transfer from the donor dye to the acceptor dye is 50% efficient.

Question 13

FRET Detection Measurements

Answer 13

1. Emission measurement - Excitation of the donor and detection of the light emitted by either the donor and/or the acceptor in the presence of the other fluorophore. When FRET occurs, the donor emission is decreased and the acceptor emission is increased.
2. Non fluorescent Dyes used as Fluorescence quenchers for FRET
3. Donor photobleaching

Question 14

Nonfluorescent Acceptor Dyes for FRET Assays

Answer 14

1. Eliminate the background fluorescence that results from direct acceptor excitation
2. QSY 7 and QSY 33 dyes efficiently quench a broad assortment of fluorescent donors, including fluoresceins, Oregon Green 488 and 514 dyes, Alexa Fluor 488 and 532 dyes and other similar fluorophores

Question 15

Donor photobleaching in FRET

Answer 15

• Measure photobleaching of donor in presence and absence of acceptor molecules
• Donor photobleaching rate is faster In absence of acceptor

Question 16

What should you be aware of during FRET measurements?

Answer 16

1. Bleed-through in excitation: i.e., when a donor is excited by the acceptor’s excitation wavelength and vice versa; and
2. Crosstalk in emission detection: i.e., when the emission of a donor also contributes to the signal measured in a setup for acceptor detection, and vice versa.

Question 17

Applications of FRET

Answer 17

1. Structure and conformation of proteins
2. Spatial distribution and assembly of protein complexes
3. Receptor/ligand interactions
4. Immunoassays
5. Probing interactions of single molecules
6. Structure and conformation of nucleic acids
7. Detection of nucleic acid hybridization
8. Primer-extension assays for detecting mutations
9. Automated DNA sequencing
10. Distribution and transport of lipids
11. Membrane fusion assays.
12. Membrane potential sensing
13. Fluorogenic protease substrates

Question 18

Considerations during FRET measurements

Answer 18

1. The concentrations of donor and acceptor fluorophores must be closely controlled.
2. The statistically highest probability of achieving fluorescence resonance energy transfer occurs when a number of acceptor molecules surround a single donor molecule.
3. Photobleaching must be eliminated because the artifact can alter the donor-to-acceptor molecular ratio, and therefore, the measured value of the resonance energy transfer process.
4. The donor fluorescence emission spectrum and the acceptor absorption spectrum should have a substantial overlap region.
5. There should be minimal direct excitation of the acceptor in the wavelength region utilized to excite the donor. A common source of error in steady state FRET microscopy measurements is the detection of donor emission with acceptor filter sets.
6. The emission wavelengths of both the donor and acceptor must coincide with the maximum sensitivity range of the detector.
7. The donor molecule must exhibit sufficiently long lifetime in order for resonance energy transfer to occur.

Question 19

Kinase Assays using FRET

Answer 19

DO THE EXERCISE FROM CLASS!

Question 20

Time-Resolved Fluorescence (TRF)

Answer 20

TRF spectroscopy provides a measure of the time dependence of fluorescence intensity after a short excitation pulse.

In TRF, pulsed light is used as the excitation source and fluorescence lifetimes are measured from the fluorescence decay signal directly.

Question 21

Why perform TRF studies?

Answer 21

1. The majority of organic dyes used for FRET have a fluorescence lifetime < 10 ns.
2. Background fluorescence from biological samples, buffer components, and plasticware also have similar lifetimes, and this can limit assay sensitivity.
3. To overcome this problem, dyes with considerably longer lifetimes have been used in time-resolved fluorescence (TRF) assays whereby the detection and measurement of assay signal is delayed until background fluorescence has dissipated

Question 22

What are the probes for Time-Resolved fluorescence

Answer 22

Chelated rare lanthanide metals

Question 23

Why do lanthanides need to be chelated?

Answer 23

1. Exist as trivalent cations

2. Have a low extinction coefficient (poor ability to absorb light) and produce a weak fluorescence.

Question 24

Properties of Chelated lanthanides

Answer 24

1. Lanthanides are excited through chelated organic compounds referred to as ‘chelates” or ‘ligands’.
2. Lanthanide chelates absorb light from the near-ultraviolet region, become excited, and give off an extremely strong fluorescence.
3. Solvents like water can quench their light-emitting properties.

Question 25

Examples of Lanthanides used

Answer 25

1. Europium 2. Dysprosium 3. Samarium 4. Turbium

Question 26

Ligands for Chelation

Answer 26

1. Calixarenes 2. Polypyridines 3. Polyaminopolycarboxylic acids 4. Beta-diketones

Question 27

How do you achieve higher than normal luminescence with lanthanide complexes?

Answer 27

Use ligands with organic chromophores with suitable photophysical properties

Question 28

What is an important value of lanthanide chelates?

Answer 28

Different lanthanide chelates exhibit different excitation and emission wavelengths, and also different lifetime constants, which allows for multiplexing of different labels.

Question 29

Unique Properties of Lanthanide chelate-based fluorescence probes

Answer 29

1. Longer fluorescence times 2. Higher quantum yield 3. Sensitivity at 10-1000 fold 4. Emission band is very sharp (10-20 nm) 5. Large stoke shifts (absorption at UV below 400 nm and emit maximally at > 550 nm) 6. Luminescence and Chemiluminescence capability 7. Low inherent background and noise level

Question 30

Fluorescence Intensity

Answer 30

Advantages - Simple, easily miniaturized Disadvantages - Sensitive to autofluorescence

Question 31

Fluorescence Polarization

Answer 31

Advantages - Simple ratiometric Disadvantages - Sensitive to autofluorescence and unstable for large ligands

Question 32

FRET

Answer 32

Advantages - Variety of probe pairs Disadvantages - Sensitive to autofluorescence and short distance (<5-10 nm)

Question 33

TRF

Answer 33

Advantages - Robust, insensitive to autofluorescence, and Long distance (5-10 nm) Disadvantages - Complex labels, limited choice of probes

Question 34

Advantages of Dissociation-Enhanced Lanthanide Fluorescent Immunoassays (DELFIA) Technology

Answer 34

1. Multiplexing 2. Excellent flexibility 3. High sensitivity - femtogram-level detection 4. Wide dynamic range 5. Low background: large Stokes' shift and time-resolved measurement 6. Superior stability

Question 35

Importance of Long fluorescent decay times and large Stokes shift

Answer 35

They allow delayed signal measurement at a wavelength with little background interference. Lanthanides emit a stable fluorescent signal that exhibits a sharp emission peak and high fluorescence intensity

Question 36

5 arrangements of DELFIA

Answer 36

Check slides

Question 37

Alpha Solutions

Answer 37

AlphaLISA - Type of ELISA assay, used when working with large number of samples from various matrix, serum or plasma AlphaLISA SureFire Ultra and AlphaScreen SureFire - This assay used when studying pathways or phosphorylated protein in whole cells or studying endogenous receptors AlphaScreen - Used when studying protein-protein interactions, fusion-tagged proteins, phosphorylated peptide or samples in a simple matrix Alpha Toolbox - Provides reagents to create your own assay (there is no off-the-shelf kit)

Question 38

AlphaLISA Technology

Answer 38

1. Bead-based non-radioactive assay that uses luminescent oxygen-channeling chemistry 2. It allows for the detection of molecules of interest in a homogeneous, no-wash format. 3. A biotinylated anti-analyte antibody binds to streptavidin-coated Donor Beads while another anti-analyte antibody is conjugated directly to AlphaLISA acceptor beads. 4. Both antibodies bind to the analyte, when present, bringing the donor and acceptor beads in the close proximity of each other. 5. Upon excitation at 680 nm, the donor beads emit singlet oxygen molecules that travel in solution to activate the acceptor beads, which then emit a sharp peak of light at 615 nm. 6. This light emission can then be detected on an Alpha-enabled reader.

Question 39

Radioactivity

Answer 39

Radioactivity is the natural and spontaneous process by which the unstable atoms of an isotope of an element transform or decay to a different state and emit or radiate excess energy in the form of particles or waves. These emissions are energetic enough to ionize atoms and are called ionizing radiation.

Question 40

Gamma Ray

Answer 40

A gamma ray is a packet (or photon) of electromagnetic radiation emitted from the nucleus during radioactive decay and occasionally accompanying the emission of an alpha or beta particle. Gamma rays are identical in nature to other electromagnetic radiations such as light or microwaves but are of much higher energy.

Question 41

Blockers of some radioactive labels

Answer 41

Alpha - Paper Beta - Plastic Gamma - Lead Neutron - Concrete