detect and identify
Biomolecular Applications
Mithras
with
and TriStar
multimode plate readers
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
Bioanalytic
Basic information
Mithras and TriStar
FRET: CFP/YFP
Fluorescence Polarisation: nuclear
receptor
HTRF®: multiplexing
BRET: receptor dimerisation
PathHunter™: GPCR
Biomolecular Interactions, Manchester, June 2009
Basics
Bioanalytic
Luminescence
„… is light that usually occurs at low
temperatures, and is thus a form of
cold body radiation. It can be caused
by chemical reactions, electrical
energy, …“ (Wikipedia)
Biomolecular Interactions, Manchester, June 2009
Basics
Bioanalytic
types of luminescence:
Bioluminescence
Chemo/Chemiluminescence
Mechanoluminescence
Thermoluminescence
Radioluminescence
Photoluminescence
Phosphorescence
Fluorescence
Biomolecular Interactions, Manchester, June 2009
Basics
Bioanalytic
Fluorescence
„… is a luminescence that is mostly found as
an optical phenomenon in cold bodies, in
which the molecular absorption of a photon
triggers the emission of a photon with a
longer (less energetic) wavelength. The
energy difference between the absorbed and
emitted photons ends up as molecular
rotations, vibrations or heat.“ (Wikipedia)
Biomolecular Interactions, Manchester, June 2009
Basics
Fluorescence: Jablonski diagram
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
Basics
Fluorescence:
Stokes shift
λem stays
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
Selecting Filters
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
Selecting filters
Bioanalytic
100
90
80
70
60
50
40
30
20
10
0
400
450
Biomolecular Interactions, Manchester, June 2009
500
550
600
Selecting filters
Bioanalytic
100
90
80
70
60
50
40
30
20
10
0
400
450
Biomolecular Interactions, Manchester, June 2009
500
550
600
Selecting filters
Bioanalytic
Overlap

100
90
80
70
60
50
40
30
20
10
0
400
420
440
460
480
Biomolecular Interactions, Manchester, June 2009
500
520
540
560
580
600
Selecting filters
Bioanalytic
Excitation and emission filter pairs must
not to overlap with their transmission
spectra!
100
90
80
70
60
50
40
30
20
10
0
400
450
Biomolecular Interactions, Manchester, June 2009
500
550
600
Selecting filters
Berthold Filter Data
Base
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
Selecting filters
Bioanalytic
Web tools
Invitrogen Spectra Viewer
http://www.invitrogen.com/site/us/en/home/support/
Research-Tools/Fluorescence-SpectraViewer.html
Omega Curv-O-matic
https://www.omegafilters.com/curvo2/index.php
Fluorophores.org
http://www.analytchem.tugraz.at/fluorophores/Browse
Substance.php
Biomolecular Interactions, Manchester, June 2009
Mithras & Tristar
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
Mithras
&
Luminescence
BRET/BRET²
AlphaScreen™
Absorbance
Prompt Fluoresc.
FRET
Fluor. Polarisation
Time-Resolved Fl.
HTRF®
Biomolecular Interactions, Manchester, June 2009
TriStar
Bioanalytic
Luminescence
BRET/BRET²
Absorbance
Prompt Fluoresc.
FRET
Mithras & TriStar
DOPS
Dedicated Optical Path System
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
Mithras & TriStar
Bioanalytic
Patented cross-talk reduction device
Aperture wheel
10-6 in black plates
Biomolecular Interactions, Manchester, June 2009
Mithras & TriStar
Bioanalytic
Photon Counting PMT operation
Stringent PMT detector selection
tim e [sec]
Biomolecular Interactions, Manchester, June 2009
10
50
0
90
00
45
00
15
00
30
00
Glow ell1
9000000
8000000
7000000
6000000
5000000
4000000
3000000
2000000
1000000
0
0
RLU
Glow ell2
ave 7836035 sd 13137 cv 0.2%
829406
1096
0.1%
89071
322
0.4%
75
00
Glow ell3
60
00
Glowell Mithras
Mithras & TriStar
Reagent Injectors: JET technology
Teflon bellows
No friction
Cell-friendly
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
00
:0
0:
00
00
0
:0
0:
1
0
00
:0 0
0:
00 200
:0
0:
00 300
:0
0:
40
00
0
:0
0:
5
0
00
:0 0
0:
00 600
:0
0:
70
00
0
:0
0:
8
0
00
:0 0
0:
9
0
00
:0 0
1:
00 000
:0
1:
10
00
0
:0
1:
00 200
:0
1:
00 300
:0
1:
40
00
0
:0
1:
5
0
00
:0 0
1:
6
0
00
:0 0
1:
00 700
:0
1:
80
00
0
:0
1:
00 900
:0
2:
00 000
:0
2:
00 100
:0
2:
20
00
0
:0
2:
3
0
00
:0 0
2:
00 400
:0
2:
50
00
0
:0
2:
6
0
00
:0 0
2:
00 700
:0
2:
00 800
:0
2:
90
00
:0 0
3:
00
0
RLU
Mithras & TriStar
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
Reagent Injectors: JET technology
Fast and efficient mixing
Berthold performance test
100000
90000
150 msec after injection
80000
70000
60000
50000
40000
30000
20000
10000
0
time [sec]
Mithras & TriStar
Reagent Injectors: JET technology
Highest precision and accuracy
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
FRET CFP/YFP
Bioanalytic
Spectral overlap of
donor em and
acceptor ex spectra
Close proximity
1 – 10 nm
Förster distance
Biomolecular Interactions, Manchester, June 2009
FRET CFP/YFP
2 readings
Ratio calculation
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
FRET CFP/YFP
JBC paper, June 2006, Nikolaev et al.
Partial agonism at the α2A adrenergic
receptor
Heterotrimers
Gi type
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
FRET CFP/YFP
Bioanalytic
cAMP monitoring with Epac2
Biomolecular Interactions, Manchester, June 2009
JBC, June 2006, Nikolaev et al.
and
JBC, June 2004, Nikolaev et al.
FRET CFP/YFP
Bioanalytic
cAMP monitoring with Epac2
Biomolecular Interactions, Manchester, June 2009
JBC, June 2006, Nikolaev et al.
and
JBC, June 2004, Nikolaev et al.
FRET CFP/YFP
Bioanalytic
cAMP monitoring with Epac2
cAMP dose-response
JBC, June 2006, Nikolaev et al.
Biomolecular Interactions, Manchester, June 2009
FRET CFP/YFP
Bioanalytic
cAMP monitoring with Epac2
NE (norepinephrine): full agonist
JBC, June 2006, Nikolaev et al.
Biomolecular Interactions, Manchester, June 2009
Fluor. Polarisation
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
Fluor. Polarisation
Excited state
has a lifetime
(ns range)
During this time
the unbound
fluorophore
rotates and the
polarisation of
the emitted
light differs
from the
excitation plane
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
Fluor. Polarisation
1st reading: parallel orientation
2nd reading perpendicular
orientation
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
FP Receptor-Ligand Binding
Bioanalytic
ERα
50 nM FITC tagged short peptide bearing the
nuclear receptor box 2-binding motif of the
co-activator protein SRC-1
Titration of receptor
Biomolecular Interactions, Manchester, June 2009
FP Receptor-Ligand Binding
150
mP
100
50
0
1
10
100
1000
Recombinant ER concentration (nM)
Scatchard analyis: Kd 7.5 nM
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
HTRF®
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
HTRF®
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
HTRF® multiplexing IP1 and cAMP
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
HTRF® multiplexing IP1 and cAMP
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
HTRF® multiplexing IP1 and cAMP
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
Bio- / Chemi-Luminescence
Most efficient light collection
Most efficient avoidance of crosstalk
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
Chemiluminescence
Bioanalytic
Enzyme Fragment Complementation
ß-Galactosidase fragment fused to receptor (“Prolink
tag”)
Enzyme fused to ß-Arrestin
Biomolecular Interactions, Manchester, June 2009
Kaiser et al., Greiner
Chemiluminescence
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
Kaiser et al., Greiner
BRET
Substrate: Coelenterazine 1 – 5 µM
Biomolecular Interactions, Manchester, June 2009
Bioanalytic
BRET: receptor dimerisation
Bioanalytic
HEK293 transfected with cDNA encoding CCR4
and CXCR5 receptors fused to hRluc and YFP
Biomolecular Interactions, Manchester, June 2009
Issad, Jockers, Marullo, Inst. Cochin
BRET: receptor dynamics
Bioanalytic
HEK cells were cotransfected with the
cDNA coding for IRRluc and YFPPTP1B-D181A
tyrosine
phosphatase
Biomolecular Interactions, Manchester, June 2009
Issad, Jockers, Marullo, Inst. Cochin
BRET: receptor conform. changes
Bioanalytic
Co-expression
of MT1-Rluc and
MT1-YFP
or
MT2-Rluc and
MT2-YFP
Biomolecular Interactions, Manchester, June 2009
Issad, Jockers, Marullo, Inst. Cochin
Bioanalytic
Thank you for your attention
Bernd Hutter
[email protected]
www.Berthold.com/bio
Biomolecular Interactions, Manchester, June 2009