Qdot Conjugates: Sensitive, Multicolor, Stable Fluorescence

Patricia Whaley, Ph.D. Molecular Probes Labeling and Detection Technologies Invitrogen Corporation

Outline

Quantum Dot Basics Materials Spectral Properties Microscopy Applications Immunofluorescent Cell Biology Molecular Pathology Cellular Assays Flow Cytometry In-vivo imaging Western Blotting

What Are Quantum Dots?

Highly fluorescent, nanometer-size, single crystals of semiconductor materials
25nm

655

605

585

565

525 nm

Size of the nanocrystal determines the color Size is tunable from ~2-10 nm (3%) Size distribution determines the spectral width

Qdot Conjugates are Engineered

Core Nanocrystal (CdSe) - Determines color

Inorganic Shell (ZnS) Improves brightness and stability Organic Coating - Provides water solubility and functional groups for conjugation Biomolecule -Covalently attached to polymer shell - Immuoglobulins - Streptavidin, Protein A - Receptor ligands - Oligonucleotides

15 - 18 nm

Size of Qdot Nanocrystals

QD 525

QD 565

QD 585

~4nm

~5nm

~5nm 5nm + 20 nm Gold

QD 605

QD 655

~5 x 12nm

~8 x 15nm

Images provided by Mark Ellisman, National Center for Microscopy and Imaging Research, UCSD, San Diego, CA
5

Quality CountsZStem Shows It

Quantum Dot Corp Materials

Literature Methods

Data courtesy Steve Penneycook (ORNL), James McBride, and Sandy Rosenthal (Vanderbilt)
6

Qdot Conjugates vs. Competitors

Not Created Equal: Immunosorbent Results 4500 4000 3500 RFU on Plate Reader 3000 2500 2000 1500 1000 500 0
-0.50 0.00 0.50 1.00 1.50

Qdot 605 Anti-Rabbit Lot 1 Qdot 605 Anti-Rabbit Lot 2 Competitor 600 (Fort Orange) Anti-Rabbit 10x competitor data

Clear Dose-Response

NO measurable binding
2.00 2.50

Log (Conj Conc nM)

Spectral Properties

Organic dye (FITC)

Qdot Conjugate (525)

Small Stokes shift Multiple source excitation reqd. Broad emission Poor photostability

Large Stokes shift Single-source excitation Narrow emission Excellent photostability

Excitation Spectra of Qdot Conjugates

5.0E+06 4.5E+06 Extinction Coefficient (M-1cm-1) 4.0E+06 3.5E+06 3.0E+06 2.5E+06 2.0E+06 1.5E+06 1.0E+06 5.0E+05
Qdot Qdot Qdot Qdot Qdot Qdot Qdot 525 Conjugate Absorbance 565 Conjugate Absorbance 585 Conjugate Absorbance 605 Conjugate Absorbance 655 Conjugate Absorbance 705 Conjugate Absorbance 800 Conjugate Absorbance

Organic Dyes { 0.0E+00

350 450 550 650 750 850 Wavelength (nm)

High extinction >> High brightness All colors can be excited at the same wavelength, 425DF45

Emission Spectra of Qdot Conjugates

800 705 655 605 565 525

400 500 600 700 800 900

Wavelength (nm)

Minimal (<5%) cross-talk using 20nm bandpass filters Simplified signal un-mixing >> simplified multiplex labeling

10

Excellent Brightness Provides High Sensitivity

Quantum dot Organic dye
High level Her 2/neu expression in SK-BR-3 Cells Quantum dots up to 50x brighter

Exp. Time: 0.019 seconds

1.22 seconds Low level of Her 2/neu expression in MDA-MB231 cells Quantum dots easy to detect but dye undetectable

Exp. Time: 0.44 seconds

8.12 seconds

11

Detection of More Parameters in a Single Experiment

Partial information from single color experiments

525 (Mouse) Mitochondria

565 (Rat) Tubulin

605 (Rabbit) Ki-67

655 (Human) Nuclear antigen

705 (Streptavidin) Actin

More information from every sample using Qdot Conjugates

12

Five Color Multiplexed Cell Labeling

Overlayed Pseudocolor
Full information from multiplex experiments

Wu, X., et al., Methods in Cell Biology, 75 2004.

5-color labeling with dyes would be extremely difficult. Multiplexing gives more information from a single experiment. Multiplexing gives much more information than 5 single experiments.

13

Direct Conjugates Provide Ultimate Flexibility

14

Qdot Conjugate Label Quality

HeLa cells fixed in paraformaldehyde and permeablized with Triton

Qdot 655 Conjugate

Alexa 594 conjugate

No significant differences in cytoskeletal protein labeling

15

Qdot Conjugates and Pathology

Molecular Pathology Patient diagnosis and prognosis Patient stratification for best treatment regimen Biomarkers for preclinical/clinical drug evaluation Traditional pathology loses all cellular/molecular correlations Morphological correlation rather than molecular correlation Multiple markers are becoming the norm Gene expression data Protein analysis Qdot conjugate stability, brightness and multiplex capability are ideal.

16

Obtain More Information From Each Specimen

Slice 1

Slice 2

Slice 3

Typical pathology: Single color, no relative measurements. Slices have no relative orientation. Marker information available is based on morphological correlations.

17

Pathology: See What Youve Been Missing

Courtesy of Jason Adams UCSF

18

Conventional IHC vs Fluorescence

Estrogen Receptor: monoclonal Rb clone SP1 RM-9101-S

Peroxidase -DAB

Qdot 655 Conjugate

RGB images acquired a single gray scale images at 655/20, 565/20, and 450/58 nm and merged Images courtesy of

19

Her-2 in Breast Cancer

20

Qdot Primary Conjugates

WGA Qdot 655 Conjugate Phalloidin Qdot 525 Conjugate

Ki-67 Qdot 585 Conjugate Vimentin Qdot 525 Conjugate (Clone V9)
21

Correlative Light and Electron Microscopy

Giepmans, et al. Nature Methods 2(10), 2005.

Images provided by Mark Ellisman, National Center for Microscopy and Imaging Research, UCSD, San Diego, CA

Glial fibrillar acidic protein - Qdot 655 Inositol triphosphate Receptor Qdot 525
22

Correlative Light and Electron Microscopy

Better EM labels than colloidal gold because of superior penetration

Qdot 655

Glial fibrillar acidic protein - Qdot 655 Inositol triphosphate Receptor Qdot 525

Qdot 525

23

Qdot Conjugates and Spectral Imaging

Standard color image (500-720 nm) 655 nm QD

565 nm QD

DAPI Unmixed composite Nuance image

Autofluorescence

Data Courtesy of CRI-Inc on a Nuance Camera System

You can see clearly now.

24

Photostability of Qdot Conjugates

605 605

Top panel (a-e): Nucleus labeled with Qdot conjugates and microtubules labeled with Alexa Fluor 488 Bottom panel (f-j): Nucleus labeled with Alexa Fluor 488 and microtubules labeled with Qdot conjugates Left: quantitative data showing effect of antifade medium

Wu, X., et al. Nature Biotechnology, 21(1) 2003.

25

 Conjugates for Fluorescent Labeling Qdot

Extremely Extremely bright bright for for sensitive sensitive detection detection High High photostability photostability provides: provides: Ability Ability to to monitor monitor signal signal over over long long periods periods of of time time Ease Ease of of use use (time (time for for focusing focusing and and image image collection) collection) Enables Enables pathology pathology and and live live cell cell imaging imaging applications applications Narrow Narrow emission emission peaks peaks for for simple simple multiplexing multiplexing Easily Easily illuminated illuminated by by many many excitation excitation sources sources Easily Easily conjugated conjugated to to a a variety variety of of biomolecules biomolecules Key Key issues: issues: Fixation Fixation Protocol, Protocol, Filter Filter Selection, Selection, PAP PAP Pen Pen Quenching, Quenching, Photobrightening Photobrightening

More data faster

26

Cellular Assays

27

Nuclear Translocation Assay

Nuclear expression of phosphorylated Erk in starved HeLa cells following EGF stimulation
1800 RFU (Mean nuclear intensity) 1600 1400 1200 1000 800 600 400 200 0 Unstimulated EGF (100ng/ml)

Unstimulated

EGF (10 min)

28

Plate-based Cellular Immunoassays Benefits

High information content in high throughput Lower cost and simpler instrumentation requirements Plate readers are readily available in all target labs More valid biology More flexibility in assay configuration More controls or reference markers may be included Lower level of expertise required Instant data reduction Image analysis and storage not required Label reagents can be transferred to tissue characterization in preclinical models

29

Qdot Qdot Conjugate Conjugate Labeling Labeling of of MAP MAP Kinase Kinase Pathway Pathway in in 3T3 3T3 cells cells
Time 0 pErk pAkt pMEK NSB
250 200 150 RFU 100 50 0 0 10 25 40 70 115 Time in PDGF (50ng/ml) MEK 1 pAkt pErk

10

10 25 25

40 40

70

70 115

pErk

pAkt

Serum starved cells were stimulated with 50 ng/ml PDGF for the times indicated. Cell (left) were stimulated for 25 min for fixation.

pMEK
30

Phospho-Erk Assay Results

Min 0 10 30 40 60 80 160
Mean RFU, (n=3)

pan Erk 605 Qdot

pErk 565 Qdot

Induction of phosphorylated Erk by PDGF in NIH3T3 cells

1600 1400 1200

pERK_PDGFpERK_PDGF+ panERK_PDGFpanERK_PDGF+

20

1000 800 600 400 200 0 0 50 100 Minute 150 200

At left: Images of the same set of wells acquired at two colors assaying pan Erk and phospho
Erk. The red circle represents the area used for measuring mean intensity in each well. At right: Results plotted showing time course of PDGF induced phosphorylation of Erk

Z values at 20 through 80 minutes are 0.63-0.77

31

More Controls Leads to More Robust Data

Kodak MM2000 gel imager

N o r m a liz e d R F U R a t io o f p E r k t o T u b u lin 10 8 6 4 2 0 0.1 1 10 100 Log PDGF (ng/mL)
3T3

BMG PolarStar Plate Reader

Norm alized RFU Ratio of pErk to Tubulin 10 9 8 7 6 5 4 3 2 1 0 0.1

3T3

10

100

Log PDGF/EGF (ng/mL)

p42/44 MAPK phosphorylation was indexed to tubulin but can be normalized to pan protein expression or wheat germ agglutinin (Qdot WGA Conjugate) expression. Multiplexing with Qdot Conjugates allows extensive information and referencing in a single well readout.
32

Respiratory Syncitial Virus Progression

Cellular infection

1 hour after infection

1 day after infection

Syncitia formation

2 days after infection

3 days after infection

E. Bentzen, Nano Letters, 5 2005.

33

Dose-Response for Viral Infection in Culture

Whole well F-Protein cell intensity vs initial infection load Data collected on a Bio-Tek Synergy HT Ex:250 nm Em:598+/-18 nm Qdot 605 Streptavidin Conjugate Biotinylated anti-RSV F-Protein Detection limit of 35-50 PFU in 24 hours

Progression can be monitored by image analysis or by quantitative analysis of the cell population. Faster and easier to look at the whole population.
34

Qtracker Cell Labeling Kits

Lagerholm, B.C., et al., Nano Letters, 4(10) 2004.

35

Three Color Qtracker Cell Labeling

10x

3T3(green), HeLa(red), and U188(white) cells labeled with Qtracker 565, 655, and 705 respectively. Co-cultured in 8-well chambers for 24 hrs. Images captured with a Leica Confocal microscope (ex. = 488nm).

36

Multiplexed Cellular Assays

CHO-655
2500 CHO 2000 Number of Cells 1500 1000 500 0 0 Day 4 SH-SY5Y

SH-SY5Y-705
2500 2000 Number of Cells 1500 1000 500 0 0

Mixed

Single cell proliferation over 4 days

Mixed cell proliferation over 4 days

CHO SH-SY5Y

4 Day

TTP Acumen Explorer for single-excitation, multiplexed cellular analysis. Real-time proliferation readouts from multiple cell-lines within a single well. Other multiplexed cellular readouts possible (Internalization, Calcium, etc.)

37

Cell Fusion Assays and Stem Cells

Endothelial Progenitor Cells Qtracker 565 H9C2 Cells (Cardiac Lineage) Qtracker 655 Cell Fusion Rate: Both colors, one cell 0.50 +/- 0.23% EPC committed to Cardiac Lineage 30-50% Transdifferentiation Conclusion: Cell Fusion cannot account for differentiation behavior

Murasawa, et al. Arterioscler Thromb Vasc Bio, 25(7) 2005, 1388-1394

38

Multiplexed Cellular Assays

Qtracker Cell Labeling Kits are non-toxic Analysis of phenotype, metabolism, proliferation, differentiation Qtracker Reagents do not transfer between cells Qtracker Reagents are passed to daughter cells for 6-8 generations typically Ideal tools for studying cell-cell interactions Ideal tools for tracking cell fate in living systems

39

Qdot Conjugates in Cytometry

Flexible excitation Usable with all common platforms Perfect for single laser, multicolor systems High brightness Comparable to or better than best dye molecules Very low cross-talk No or minimal compensation required from single laser. Improving platform QDC R&D producing ongoing improvements to brightness, width, and NSB. Unlimited colors available 6 colors from 525-800 nm with < 5% cross-talk Photostability allows for imaging and resorting

40

Flow Cytometry: Qdot Streptavidin Sampler Kit

CD4-biotin + Streptavidin Sampler Kit on human PBMCs, 405 nm excitation
Qdot 525 streptavidin 525/20 Qdot 605 streptavidin 605/20

Qdot 565 streptavidin 565/20

Qdot 655 streptavidin 655/20

Qdot 585 streptavidin 585/20

Qdot 705 streptavidin 695/40

41

Single Laser, Multicolor Flow Without Compensation

Qdot 585 CD8

Qdot 655 CD4

PBMCs + direct conjugate cocktail Modified BD FACScan: FL1: 530/30 FL2: 585/42 FL3: 620 LP Qdot 585 CD8 Run without compensation

Qdot 525 CD3 Reagent Qdot 525 crystal Qdot 585 crystal Qdot 655

Intensity off 488 nm ~ Fluorescein ~ 50% RPE ~ RPE/Cy5

Spectral Overlap 2% into FL2 11% into FL3 (>620 nm) 0.2% into FL2

42

Clean Spectral Signals from Qdot Conjugates

17 colors with manageable compensation

Other dyes dont cross-over into Qdot Conjugate channels Qdot Conjugates dont cross-over into other dye channels substantially (in spite of broad excitation)
Perfetto, S.P., Chattopadhyay, P. K., Roederer, M.; Nature Reviews Immunology; V.4 (2004); 648-655.

43

4 Qdot Tetramer Reagents

More information per sample possible Alexa Fluor680 CD8 Phenotyping & Activation in a single sample context

CMV
(Qdot 705 streptavidin) (Qdot 705)

Gag
(Qdot 655 streptavidin) (Qdot 655)

Streptavidin Conjugates used with Biotin-MHCPeptide complexes to elucidate T-cell specificity

EBV
(Qdot 545 streptavidin) (Qdot 545)

Nef
(Qdot 605 streptavidin) (Qdot 605)

Data courtesy Stephen DeRosa (FHCRC) and Mario Roederer (NIH-VRC)

44

LSR II: Trigon / Octagon Configurations

440/40

72 0
xxx /xx

P 505LP xxx LP

UV nm Laser

/20

52 5/ 50

515/10

63

5L

695/60

532 nm Laser

550L

65 5

/20

63

5L

P
5 55

660/20

P 505LP

690L P

720/20

60

5/

20

633 nm Laser

405 nm Laser

525/50

57

0L

735L P

787/42

67

0/

14

5 05L P 7 30L P 7 60L P

68

5L

P
5 45

58 5 /4 2

488 nm Laser

LP

488/10

78 0/ 60

585 /4 2
58 5 /4 2

LP

78 7/ 42

440/50

45

5-Color Stain: Violet Excitation

Pacific OrangeTM CD8 Qdot 605 CD19 CD3-b + Qdot 655 SA CD56-b + Qdot 705 SA CD19-b + Qdot 605 SA Pacific Orange CD8 / Pacific Blue CD4 Pacific BlueTM CD4 Qdot 655 CD3

Qdot 705 CD56

Qdot 705 CD56

Pacific Blue CD4

Pacific Orange CD8

Qdot 705 CD56

Qdot 655 CD3

46

Summary
Current Qdot nanocrystals are comparable to best organic dyes in intensity. Narrow emission spectra of the Qdot nanocrystals result in very low cross-talk. Selected Qdot reagents exhibit minimal spectral overlap Qdot reagents can be multiplexed for multicolor analysis in single- and multi-laser systems Current solutions available: Streptavidin and anti-immunoglobulin conjugates Conjugation kits Reactive Qdot nanocrystals Custom conjugation services

47

Direct Conjugates Give Excellent Results

Chattopadyhay, et al. Methods in Molecular Biology (in press)

At the right titration level

48

Importance of Optimal Titration and Clone

Signal of Positives and Negatives (Qdot 655 Leu3a/RPA-T4)
10000

FL3-H Pop Median

1000

Leu3a Positives Leu3a Negatives RPA-T4 Positives RPA-T4 Negatives

100

10

1 0.1 1 10 100

Concentration (nM)

PBMC stained with Qdot 655 Leu3a/RPA-T4 Conjugate Washed 2x with HBSS. 100 ul staining with 106 cells. High affinity clones give high quality products.
49

Direct Antibody Conjugate Comparison

Qdot 655 vs PE-Cy5 Antibody Conjugates

CD3

CD4

CD8 Qdot 655 Antibody Conjugate PE-Cy5 Antibody Conjugate

50

Current (488 based) and Future Performance

Intensity

Crosstalk

Potential Crosstalk
<1%

655

Equal to PE-Cy5

0.2% into FL2

1% PE-Cy5

585

~1/2 PE signal

11 % into FL3 (620 LP) 52% PE

<1 % (on standard 650 LP FL3)

525

Equal to FITC

2% into FL2 14% A488 23% FITC

<1%

51

Single Laser, Multicolor Flow Without Compensation

Buffy coat stained Direct Qdot Conjugate reagent cocktail Lysed/Washed 2x 488 ex FACScan 530/30 FL1 585/42 FL2 620 LP FL3 UNCOMPENSATED Stock 650 LP would have substantially lower FL2/3 cross-talk
CD3 CD8
52

CD4 CD8

Flow Summary

Current Qdot materials are comparable to best dyes in intensity. The narrow emission spectra of the Qdot materials result in very low cross-talk. The Qdot channels off the violet laser are free of compensation. There are significant benefits to using Qdot materials in multicolor single-laser analysis. Current solutions available: Conjugation Kits Streptavidin Conjugates Reactive Qdot Nanocrystals Custom Conjugation Services

53

In vivo Imaging

Ballou, B. et al., Bioconjugate Chemistry, 15(1) 2004.

Qdot nanocrystal materials can be imaged at scales from centimeters to nanometers.

54

Real-time Receptor Ligand Tracking

EGF Receptor Internalization

Qdot 605-EGF conjugate (erbB1) erbB3-Citrine EGF-Qdot Conjugate cointernalizes with ErbB2 Novel retrograde transport mechanism via filopodia EGFR homodimerizes with erbB2 but erbB3

Photostability allows unprecedented real-time continuous monitoring of receptor-molecule (ligand, drug) dynamics
Lidke, D. et. al. Nature Biotechnology 22 (20), 2004

4.5 sec/frame; 100 frames Sequential confocal scans

55

Multiphoton Imaging Vascular Imaging in Ovary

FITC - Dextran

Qdot ITK Carboxyl QDs Single MP slice

Qdot ITK Carboxyl QDs 250 m image stack

Larson, et.al., Science 300(5624) 2003.

Bright - Very high S/B - Excellent discrimination from auto-fluorescence - Fine structural and dynamic information can be obtained

Noninvasive - Imaging through skin - High contrast angiography Stable - No toxicity observed after venous injection

56

In-vivo Vascular Imaging

Chick embryo venous injection at increasing resolution Bright signal allows highly detailed vascular analysis Red colors allow deeper, higher resolution imaging than dyes

Courtesy of Greg Fisher, Byron Ballou and Alan Waggoner, Carnegie Mellon University

Long circulation time allows detailed vascular imaging. Also useful for marking vascular structures in tissue sections.

57

Zebrafish with Qdot Conjugates

Whole Mount IHC from in-vivo Acetylated Tubulin axons Qdot 605 SAv Conj. vascular

Rieger, et al. Dev Dyn 18, 2005.

58

Tracking cells by fluorescence and luminescence

C57B1/6 Mice B16F10 luc cells Loaded with Qtracker 705 Cell Labeling Kit 2x106 cells injected via tail vein Cell fate monitored for 24 hours. Fluorescence tracks bioluminescence results until colony formation.
Xenogen IVIS Expt Courtesy Steve Smith

Results like bioluminescence without transformed cells

59

In-vivo imaging across the continuum

Molecular Targeting
Non-targeted Intravascular Cell Surface Interstitial Intracellular Metabolic Alexa Fluors Quantum Dots Alexa Fluors Quantum Dots

Cellular Tracking
Passive Proliferation Quantum Dots Alexa Fluors Uptake Metabolism Reporter Systems

Post-mortem analysis
Physiology Quantum Dots Alexa Fluors Molecular

60

Live Cell and Animal Imaging

Image noninvasively, then follow up at higher resolution Compatible with GFP imaging Imaging at greater depth and resolution Infrared materials can be imaged through skin and other tissues effectively Repeated imaging without repeated dosing. Longitudinal imaging from in-vivo to intravital to post-mortem to electron microscopy.

61

Qdot Western Blotting products

Actin Vimentin GAPDH Rat Tissue Lysate Blot

62

Western Analysis with Qdot Conjugates

Sensitivities comparable or better than reported chemiluminescence levels. NO FILM, NO DARKROOM, NO RUSH. Simple multicolor detection without dedicated instrument Chemiluminescence imaging systems Gel Documentation systems Trans-illuminator and color camera with anti-haze filter No stripping and reprobing required Faster experiments More reliable data More reliable quantitative analysis 2-3 orders of magnitude linear dynamic range with single exposure Extended exposures extend dynamic range to 4-5 orders of magnitude

63

More Sensitive than the Gold Standard ECL

Detection Limit Qdot 655 Conjugate Kodak Imager 100 pg

ECL Detection Kodak Imager ECL Detection HyperfilmPlus 5 min 12.5 6.25 3.1 1.6 0.8 0.4 0.2 0.1 0.05 (ng)

1600 pg

400 pg

Purified protein dilution series with identical antibodies Qdot Conjugates deliver sensitivity dramatically higher than ECL Reagents even under the optimal film-based detection. Combination with Millipore Immobilon-FL Transfer Membrane provides highest sensitivity.

64

Qdot Western Blots Can be Stored for Months

Day 2: 40 pg detection

3 months later: 150 pg detection Stored in TBS

Stored in TBS, the Qdot Western Blots retain signal for months. Stored dry, they may retain signal even longer. Plenty of time to get imaging conditions optimized for publication.

65

Sensitive Quantitative Western Analysis

Pure GSTGoat anti-GSTQdot 655 anti-Goat Conjugate
Detection limit 4-8 pg GST Fluorescence detection with chemiluminescent sensitivity Stability for repeated analysis

125 pg

62 pg

31 pg

500 pg

250 pg

16 pg

8 pg

Images acquired with a KODAK Image Station 2000MM Multimodal Imaging System
Secondary Detection of GST

4 pg

1 ng

1000 Mean Intensity 800 600 400 200 0 0

R2 = 0.9951

Broad linear range Progressive exposures extend range 100 fold linear range for each exposure

200 400 pg / lane GST

600

66

Useful in Protein Expression and Purification

GST-cMyc GST-HA HA detected with a primary antibody followed with Qdot 605 anti-Rabbit conjugate. GST detected with Qdot 565 anti-GST conjugate c-Myc detected with a primary antibody followed with Qdot 705 anti-Mouse conjugate. Multicolor analysis allows simultaneous measurement of protein and fusion domains. Single blot analysis eliminates questions of band alignment between multiple blots. Qdot conjugates allow 3 or 4 color analysis of overlapping bands with simple filters.

67

Simple, Fast and Economical

ECL on Film Transfer (1 hr) | Block (1 hr) | Primary (1 hr) | Rinse (0.25 hr) | Secondary (1 hr) | Rinse (0.25 hr) | Substrate, Film, Develop (1 hr) | Substrate, Film, Develop (1 hr) | Substrate, Film, Develop (1 hr) Qdot Conjugate Transfer (1 hr) | Block (1 hr) | Primary (1 hr) | Rinse (0.25 hr) | Secondary (1 hr) | Rinse and Image 3x (0.25 hr) Save hours in the darkroom with Qdot Western Blotting detection

68

Simple, Fast and Cost Effective

Detection Method
ECL Advance

Product Number

Antibody Dilution Cost per blot

(GE-Amersham)

RPN2138

1:25000

$35.45

ECL Plus

(GE-Amersham)

RPN2132

1:5000

$37.05

ECL

(GE-Amersham)

RPN2108

1:5000

$31.05

Qdot Conjugate Qdot Conjugate

1100-2 1100-2

1:1000 1:2000

$28.03 $19.28

69

Useful in Cell Signaling Research

Phosphoryation of Akt and Erk in A431 Cells Stimulated by EGF (25ng/mL) This is a typical cell-signalling model system.
phospho Akt phospho ERK 1/2

pan Akt Qdot 705 pan ERK 1/2

Qdot 605

Akt ERK1/2 0 10 20 40 60 (min)

Ornberg, et al., Nature Methods 2(1) 2005, 79-81

Color value indicates ratio of modified to total protein This is a typical class of experiment in cell-signalling research. Analysis of multiple bands allows single experiment with rich content. The alternative experiment would take several days with standard methods.

70

Qdot Western Blotting Benefits

Simple Fluorescence imaged directly, no substrate addition No dependence on time (CL) or photostability issues (dyes) Robust, stable signals allow repeated imaging Imaging can be done on gel imagers, CCD imagers, and laser scannersvery flexible Quantitative Linear quantitative range over 2.5 orders of magnitude Stable signal ensures reliable measurements Sensitivity as good as best reported chemiluminescence methods Multiplexed No stripping and reprobing to detect multiple bands Single source excitation with emission filters ensures reliable signal ratios

71

Conclusions
Qdot Conjugates provide substantial benefits in detection Ultimate in photostability Sensitivity rivals or exceeds the best methods Multiplexing capability dramatically simplified Wide variety of available products ensures application needs are met Distinct product lines appropriate for many applications Microscopy Flow Cytometry Live cell/live animal imaging Western Blotting Immunoassays Reactive Qdot nanocrystal materials available for your chemistry (Innovators Tool Kit Quantum Dots) Organic Carboxyl Amino(PEG)

72

Independent Accolades
Science magazines Top 10 Scientific Breakthroughs of 2003. [Quantum dot bio-imaging is]the most exciting new technique to emerge from the collaboration of physicists and biologists. Forbes/Wolfe Nanotech Reports Top 5 Breakthroughs of 2003. Number 1: In vivo labeling with quantum dots LARTA Nano Republic Conference 2003. Most promising innovation award. Small Times magazines 2003 Researcher of the Year. Quantum Dot founder Paul Alivisatos. 2004 Fortune Cool Companies winner.

73

PublicationsInvitrogen Materials Are the Standard

~230 peer reviewed publications (Biological Apps since 1998) 92 used QDC Materials (2003-2005) PathologyFluorescence/EM/FISH Live Cell Microscopy (dynamics) Single Molecule Analysis FRET Arrays Microfluidics/Patterning Pathogen Detection And many more 2 used competitor materials (reporting quenching) Others used self-fabricated materials (great ideas) Large number of review articles Quantum DotInvitrogen Nanocrystal Technologies materials ensure consistency, support, and optimal performance every time.

74

Collaborators

Mark Ellisman Alan Waggoner Paul Wylie Watt Webb Mario Roederer

NCMIR-UCSD Carnegie Mellon University TTP Labtech Cornell University NIH-Vaccine Research Center

75