Near infrared nanocrystalbased quantitative protein arrays

1
Near infrared nanocrystal-based quantitative
protein arrays

• EP-S19T03 Ursula Klingmüller

2
Overview
EpoR ? JAK/STAT, MAPK IL-6R ? JAK/STAT TGFß ? SMAD
activate cell signalling ?
Detection and quantification on immunoblot and
protein microarray
? detect signals
Normalization and calibration of the
data Evaluation of pathway Interactions under
normal and disease conditions
process data ?
EP-S19T03
3
Specific aims

• Systems biology approaches time-dependent
  measurements
• Information on post-translational modification
  phosphorylation
• Multiplexing analysis of different molecules
  simultaneously
• High sensitivity and specificity choice of
  antibodies
• Microarrays - increase in throughput larger
  amount of data from the same amount of sample
• Small error established protocol
• (Generation of absolute numbers molecules per
  cell)

EP-S19T03
4
Quantitative immunoblotting timecourses of
signal activation
timecourse blot picture
p-erk1/2
EP-S19T03 Sophia Derdak
5
Capture Protein Microarrays
Secondary detection fluorescence labelled
Detection phosphorylation state
independent Capture phosphorylation state
independent
Secondary detection fluorescence labelled
Detection phosphorylation state
independent Capture phosphorylation state
dependent


total amount of protein
phorphorylated portion of protein
Array setup
EP-S19T03 Ulrike Korf
6
Capture Protein Microarrays
microarray
p-erk1/2
EP-S19T03 Sophia Derdak, Ulrike Korf
7
Capture Protein Microarrays
microarray
p-erk1/2
EP-S19T03 Sophia Derdak, Ulrike Korf
8
Luminescent nanocrystals in NIR
Which nanocrystals? semiconductor nanocrystals
with band gaps between 1.5 and 1.8 eV CdTe,
CdHgSe, InP

• Why nanocrystals?
• lower quantum yields than organic dyes
• little or no photobleaching
• narrow emission peaks
• broad band excitation

Phase transfer into aequous solution
H2N
NH3
H3N
NH2
NH2
H2N
Coupling to proteins
NH2
NH-OC-
Y
EP-S19T03 Thomas Nann
9
Data Management quantmod
dilution series quality control
antibody combinations file
slide description file
antibody pairs intensity object
array design file
dilution series object
slide intensity files
protein concentrations
calibration matrix file
EP-S19T03 Achim Tresch
10
Milestones
11
Cooperations within the NGFN
Diseases of the nervous system
Cardiovascular diseases
Diseases due to environmental factors
Systematic-methodologic platforms PAB-S06T04
Stefan Dübel PBF-S19T10 Tim Beissbarth PEG-S19
T03, PRN-S19T04 Jörg Hoheisl
Explorative projects EP-S19T01 Anthony
Ho EP-S19T04 Ralf Bischoff
Cancer PRN-S19T03 Holger Sültmann N2KR-S30T04
Martin Eilers N1KR-S19T25 Günter Hämmerling
Infection and inflammation
12
(No Transcript)
13
Near Infrared Detection Nanocrystals
Helle CdHgSe NCs
Länge 10 20 nm Breite
2 10 nm Schale 2
20 nm
EP-S19T03 Thomas Nann
14
Lumineszierende Nanokristalle im NIR
Warum Nanokristalle?
Welche Nanokristalle?

• Lum. Nanokristalle haben i.d.R. niedrigere
  Quantenausbeuten (QY) als organische Farbstoffe,
  aber...
• sie zeigen kaum Photobleaching
• besitzen schmale Emissionsspektren
• lassen sich breitbandig bis ins UV anregen

Halbleiter-Nanokristalle mit Bandlücken band gap
im Bereich von ca. 1.5 1.8 eV sind
geeignet CdTe, CdHgSe, InP, ...
Phasentransfer ins Wäßrige
H2N
NH3
H3N
NH2
NH2
H2N
Kopplung an Proteine
NH2
NH-OC-
Y
15
Milestones