Transcript Document

Advantages of the
Alexa Fluor dyes in
Molecular Imaging
New Secondary Detection Tools for
Flow Cytometry and IHC,
Small Animal In Vivo Imaging Reagents
Dana Brozban
Technical Sales Specialist
Imaging and Microscopy
[email protected]
Today’s Presentation
• Focus on Key Molecular Probes Products
and Applications
• Alexa Fluor® Dyes
• Zenon ® Technology for secondary
detection
• Small Animal In Vivo Imaging (SAIVI™
Reagents)
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Desirable Characteristics of Fluorophores:
1) Emission at wavelengths that minimize problems with
autofluorescence
2) Minimal spectral overlap with other fluors (Minimal Compensation)
3) Bright (high QY and Extinction Coefficient)
4) Minimal photo bleaching for sorting and microscopy
5) Minimal non-specific cell binding
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What Does a Fluorescent Dye Look Like?
•
Rigid conjugated -electron system is generally required for
fluorescence
Phenolphthalein
Flexible, non-fluorescent
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Fluorescein
Rigid, fluorescent
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Efficiency or Total Fluorescence
e = Absorbance x Path Length
[M]
e = Extinction Coefficient, Path Length is typically 1 cm for most cuvettes, [M] = the
molar concentration
•More highly conjugated (ring structures) compounds give higher extinction
coefficient as they absorb more light.
- Benzene has an extinction coefficient of ~1000
- Ethidium Bromide has an extinction coefficient of ~ 6000
Quantum Yield = Number of Photons Emitted by the Fluorochrome divided by the
number of photons absorbed.
Another way to look at the “quality” of a fluorochrome is to use:
e x Q Yield = Efficiency or Total Fluorescence.
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Protecting the fluorescence signal
Q: What are the problem faced with standard fluorescent dyes?
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Protecting the fluorescence signal
Problem
Low signal intensity
Photobleaching
Sensitivity to environmental conditions
Solution
The Alexa Fluor® dyes: brighter, more photostable, pH insensitive
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Immuno Histochemistry Workflow
Workflow Problems
Culture Cells
Fix cells
Permeabilise
Blocking Buffer
Primary Antibody
Secondary Antibody
Nuclear Stain
Low Signal Intensity
Photobleaching
Non-specific Binding
Mount
Microscopy
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Filter Quality
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Multiplexing
Alexa Fluor® Dyes across the spectrum
Colour Selection
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♦
Brightness
♦
Photostability
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Multiplexing-The Four Main Colours
Emission
wavelengths:
350
700
Blue
400
Green
450
DAPI/UV
Alexa Fluor® 350
Coumarin, AMCA
568
488
350
647
Orange
500
Far Red
550
600
FITC
TRITC
Alexa Fluor® 488
Fluorescein (FITC)
Cy2
Alexa Fluor®
555
Rhodamine,
TAMRA, TRITC
Cy3
650
FAR RED
Alexa Fluor® 647
Cy5, APC
Alexa Fluor® 594
Texas Red, Cy3.5
Colour Selection
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Brightness
♦
Photostability
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Alexa Fluor ® 488 and fluorescein
— Alexa Fluor® 488
--- fluorescein
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Alexa Fluor ® 488 vs Fluorescein Bleaching
2x Real Time
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Alexa Fluor ® Dyes: pH Insensitive
Alexa Fluor488
Oregon
Green
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FITC
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Alexa Fluor ® Dyes – Photo Stability
Laser-scanning
cytometry
EL4 cells
biotin-anti-CD44
+ streptavidin
conjugates
Photobleaching dye
solutions in
capillary tubes
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Better Performance
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Alexa Fluor ® 647 vs. Cy5
Compare Alexa Fluor conjugates to
any other fluorescent conjugates
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Alexa Fluor® 405 / Pacific Blue®
Alexa Fluor® 405
Excitation: 405 nm
Emission max.: 421 nm
Molecular Weight: 1028 Da
Extinction Coefficient: 34,000 M-1cm-1
Pacific Blue®
Excitation: 405 nm
Emission max.: 455 nm
Molecular Weight: 339 Da
Extinction Coefficient: 46,000 M-1cm-1
•Great match for the 407 nm line of the krypton laser and the
405 and 408 violet diode lasers.
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Alexa Fluor® 405 / Pacific Blue®
anti-human
CD8
AF 405
MFI = 88
Pacific Blue
MFI = 145
•Alexa Fluor 405 Brightness- on the order of magnitude of
FITC
•Alexa Fluor 405 is purported to be less prone to
problems with non- specific binding.
•Photo-stability: 24 hour exposure, no
reduction in signal
anti-human CD45
Pacific Blue
MFI = 419
AF 405
MFI = 199
•Pacific Blue is brighter than Alexa Fluor 405.
• Pacific Blue has no compensation issues with UV
excitable dyes
• Studies have shown a decrease in signal of
Pacific Blue following fixation.
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Flow cytometry data generated at
the J. David Gladstone Institute core
flow cytometry laboratory, San
Francisco, CA, headed by Marty
Bigos, assistant Valerie Stepps
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•Therefore if you need to fix, consider using Alexa
Fluor 405
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Alexa Fluor ® 488 succinimidyl ester
SO
3
H N
2
SO
3
O
+
NH
2
CO
2
O
N
O
O
Amine reactive
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Single Conjugates
Single
Alexa
405
Alexa
405
Alexa
405
Alexa
488
Alexa
488
Alexa
488
Alexa
647
Alexa
647
Alexa
647
Single Conjugates are made by attaching small fluorescent
molecules (fluorophores) to the antibody at optimal F to P
ratio’s.
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Types of Fluors
Tandem – Fluorescence Resonance Energy Transfer (FRET)
Dyes
• Emission of one dye overlaps the excitation of a second.
• When close, energy is transferred to the second.
• The second emits a longer wavelength of light than the first.
Alexa 610
PE
Alexa 610
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Alexa 610
Alexa
750
APC
Alexa
750
Alexa
750
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Tandem Conjugates - Energy Transfer Dyes
Fluorescence Resonance Energy Transfer (FRET)
Donor
R-PE
APC
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Acceptor
Alexa Fluor® dye
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Tandem Conjugates - Energy Transfer Dyes
PE-AF 610
PE-AF 647
R-PE
APC
PE-AF 680
APC-AF 680
APC-AF 700
APC-AF 750
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R-Phycoerythrin (PE-Alexa Fluor 610)
• Large multi-subunit, globular (~240 kDa)
protein derived from algae.
• >20 chromophores per molecule
• High quantum yield (bright)
• Excitation 488 nm and 532 nm
• Emission Maximum 575 nm
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Alexa Fluor® 610 Carboxylic Acid, Succinimidyl Ester
Alexa Fluor 610 Characteristics
R-Phycoerythrin
Alexa Fluor 610
Formula Weight………… 1171.66
Absorbance Maximum… 612 nm
Extinction Coefficient….. 145,000 M-1cm-1
Emission Maximum……. 628 nm
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PE-Alexa Fluor 610
The Visible Spectrum
Ultraviolet
Infrared
Light
Light
400
500
600
Wavelength (nm)
R-Phycoerythrin, Chains: A, B, K, and L
PE-Alexa Fluor 610 an alternative to PE-Texas Red
•Energy transfer dye excited at 488,
with maximal emission at 628 nm.
•Higher quantum yield than PE-Texas Red
with lower compensation in PE channel
•Ideally suited for multicolor applications
with the superior alternative for PE-Cy5 :
PE-Alexa Fluor 647
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APC-Alexa Fluor 750
The Visible Spectrum
Ultraviolet
Infrared
Light
Light
400
500
600
Wavelength (nm)
Allophycocyanin, Chains A and B
APC-Alexa Fluor 750 Analysis
APC-Cy7 anti-human CD4
Spectra
MFI = 193
Compensation = 28%
APC Channel
1
2
750 anti-human CD4
APC-Alexa%Fluor
Compensation
• Instrument: Tritech1 modified BD FACScan™ 2
• Human peripheral blood lymphocytes
• APC-Cy7 and APC-AF750 detected through 765/20 filter, split from APC with 675
SP
Light Stability
45
40
40
35
30
25
20
28
T = 0 hrs.
21
47%
MFI = 551
T = 6.5 hrs.
16
Incr. Compensation
= 16%
15
20%
Incr.
10
5
0
CD4 APC-Cy7
CD4 APC-A750
APC Channel
Tritech, Edgewater, MD
BD, Becton Dickinson Biosciences Immunocytometry Systems, San Jose, CA
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APC-Alexa Fluor 750 vs. APC-Cy7
•
APC-Alexa Fluor 750 is perfectly
interchangeable with APC-Cy7.
•
APC-Alexa Fluor 750 is brighter than APC-Cy7
by as much as 2-fold.
•
APC-Alexa Fluor 750 exhibits roughly half the
compensation of APC-Cy7.
•
APC-Alexa Fluor 750 is significantly more
photostable than APC-Cy7.
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Antibodies and ImmunoFluorescence
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Fluorescence reagents-Labeling
Direct-Labeled Primary Antibody
• Lowest Background
• Potentially low signal due to abundance of target or dye
• Dye could affect antigen recognition site
Zenon Technology
• Brighter Signal
• Dye does NOT affect antigen recognition site
Indirect-Labeled Secondary Antibody
• Higher Background
• Brighter Signal
Tyramide Signal Amplification (TSATM)
• Higher Background
• Brightest Signal
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Fluorescence reagents-Direct
•
Simple and easy to follow protocols
•
Reactive dye is pre-measured – no need to weigh out small quantities of dye
•
Procedure (including purification) takes ~2 hours, with little hands on time
•
Three kit sizes
Alexa Fluor® Protein Labeling Kit (1 mg)
+
Alexa Fluor® Monoclonal Labeling Kit (100 ug)
New - Alexa Fluor® Microscale Protein Labeling Kit (20-100 ug)
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Fluorescence reagents-Zenon® Labeling
~ 1 μg primary
antibody
Add Zenon®
reagent at desired
molar ratio
Incubate 5
minutes
Block with nonspecific IgG
& Use directly
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Zenon labeling technology- Stability
Kinetically trapped complex formation: key to making the system work.
Fast complex formation
Mouse anti-biotin IgG1
Slow complex dissociation
Blocked mouse anti-biotin IgG1
+ Alexa Fluor 488 Zenon
Capture by BSA-biotin in microtiteer plate
+ Alexa Fluor 488 Zenon
Capture by BSA-biotin in microtiteer plate
Control IgG1
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WHY use ZENON?
•
Simple
No need to use secondary antibodies anymore
•
Speed
Zenon labeling complexes are ready to use for cell staining within 5 minutes…
•
Quantitative Labeling
100% of the primary antibody sample is labeled.
•
No Preparation
Removal of exogenous proteins such as serum albumin
from primary antibody samples is unnecessary.
•
Compatibility
Multiple Zenon One–labeled mouse antibodies can be used
in the same immunolabeling protocol.
•
Economy
A standard Zenon labeling requires only 1 µg of primary antibody
(versus 100 µg for chemical labeling).
Save time and money.
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Example 1: Triple Immunofluorescence Staining of Muntjak Cells with Zenon
• anti-nuclear mouse mAb + Alexa Fluor 350 Zenon labeling reagent
• F-actin + Alexa Fluor 488 phalloidin
• anti-tubulin mouse mAb + Alexa Fluor 568 Zenon labeling reagent
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Example 2: Switching Fluorescent Colors: Same Cell Type, Same Targets
• F-actin + Alexa Fluor 350 phalloidin
• anti-tubulin mouse mAb + Alexa Fluor 488 Zenon labeling reagent
• anti-nuclear mouse mAb + Alexa Fluor 647 Zenon labeling reagent
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Just in case you were wondering…
Muntjak
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Fluorescence reagents - Indirect
Goat anti-Rabbit IgG
Host animal in
which antibody
was raised
Animal in which
antigen was
isolated
Immunoglobin against
which antibody was
raised
We Offer a Wide Selection of Secondary Antibodies
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Tyramide Signal Amplification (TSA™)
TSA™ can provide high resolution and high S/N
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Chromosome FISH: Labeled Streptavidin vs TSA™
Streptavidin
Alexa Fluor
546
Alexa Fluor
546 TSA
Alexa Fluor 546 Dye FISH Signal: Streptavidin vs TSA
RFU
1500
1000
500
0
AF546-SA
AF546-TSA
10-fold increase in sensitivity w/ TSA™
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Why Use TSA?
3 color sequential TSA™
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Increased sensitivity
– ~100-fold compared to avidin–biotinylated
enzyme complex (ABC)
•
Generates multiple copies of
Alexa Fluor dyes at target site
•
Gives high-resolution signal amplification
•
When normal amplification methods fail
– Low abundance targets
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Immuno Histochemistry Workflow
Workflow Problems
Culture Cells
Fix cells
Permeabilise
Blocking Buffer
Primary Antibody
Secondary Antibody
Nuclear Stain
Low Signal Intensity
Photobleaching
Non-specific Binding
Mount
Microscopy
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Filter Quality
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Protecting the fluorescence signal-Antifade Reagents
Prolong® Gold
•Optimized formulation-Superior antifade with little or no
impact on initial fluorescence intensity
•Ready to use - dropper bottle
•Cures to a firm gel – no more nail polish
NEW: SlowFade® Gold - antifade mounting media which doesn’t polymerize
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Immuno Histochemistry Workflow
Workflow Problems
Culture Cells
Fix cells
Permeabilise
Blocking Buffer
Primary Antibody
Secondary Antibody
Nuclear Stain
Low Signal Intensity
Photobleaching
Non-specific Binding
Mount
Microscopy
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Filter Quality
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Increase S/N ratio – Reducing Non-specific background
Non-specific binding - any signal not related to the target
of interest, therefore increasing the background.
Problem
 Non-specific binding of dye conjugates
 Standard blocking agents don’t work
Solution
•Image-iTTM FX Signal Enhancer – non-serum based blocking reagent
Decrease background - Increase Specific Signal
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Increase S/N Ratio - Image-iTTM FX signal enhancer
BPAE Cells Stained with TMR Streptavidin
Not blocked
Blocked
Image-iTTM FX signal enhancer
decreases background fluorescence!
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Alexa Fluor® Image-iT™ FX & Image-iT™ SFX Kits
The Alexa Fluor® Image-iT™ FX Kits provide all of the dyes and
reagents you need for optimal imaging of fixed cells and tissue sections for
a more convenient price:
– Alexa Fluor® Dye conjugates for superior photostability and
brightness
– ProLong Gold antifade reagent for reduced photobleaching
independently of the color of your fluorochrome
– Image-iT FX Signal Enhancer for improved signal-to-noise ratio
- CultureWell chambered coverslips to make sample processing
more convenient.
NEW : Alexa Fluor® Image-iT™ SFX Kits = Start-Up Kits:
1. Alexa Fluor® Dye conjugates in small packaging combined
with….
2. Image-iT™ FX Signal Enhancer for superior, photostable, bright
and highly specific staining !
NEW Easier to Use Kits
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Small Animal In Vivo Imaging (SAIVI™)
DRIVERS for INVITROGEN
•
Bring our expertise in molecular biology to
develop solutions in molecular imaging
•
Develop tools to enable the acceleration of
drug discovery
•
Continue to support our customers in the
development of useful tools as they use more
in-vivo assays
The VISION for INVITROGEN
•
•
Leverage our Molecular Probes Labeling &
Detection technology in the Optical imaging space
with injectable NIR reagents and Qdot technology
– L&D
– Cell Biology
– In vivo Imaging
Leverage our other appropriate technologies (gene
regulation and delivery) into the in vivo imaging
space
COMPELLING REASONS for Optical
•
•
•
•
•
Low cost: Compared to all
Ease of use: Compared to all
High sensitivity:
– superior to anatomical MR & CT
– competitive with functional PET
Optical can enable benchtop imaging for the
PI
Quantitative Optical can displace some PET
COMPELLING REASONS for NIR Injectables
VisEn
Bioluminescence
GFP
NIR Injectables
No Background
Hi Sensitivity
Hi Light Output
Good Sensitivity
Hi Light Output
Good Sensitivity
Ease of Use
Huge Target Flexibility
Depth Penetration
Low Light Output
Labor/Cost/Time
Limited Depth
Labor/Cost/Time
Autofluorescence*
Autofluorescence*
NIR injectables can simplify and improve optical SAIVI
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Dye Technologies
Alexa Fluor Dyes
Alexa Fluor 647
Cy5, (APC)
allophycocyanin
Total fluorescence significantly higher than Cy5 conjugates, unlike Cy5, 647 has little
change in fluorescence when conjugated to most proteins, nucleic acids, thus greater
total fluorescence at the same degree of labeling.
Alexa Fluor 660
Cy5.5, APC
Good spectral separation from orange fluoreophores. Efficiently excited by 633 nm
and 647 nm laser lines
Alexa Fluor 680
NA
Highly recommended for use int the LI-COR Odyssey instrument for their “in-cell”
westerns
Alexa Fluor 750
Cy7
Longest-wavelength Alexa Fluor commercially available
Fluorescent Nanocrystals.
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SAIVI TM Imaging Reagents
CORE TECHNOLOGY
CUSTOMER TOOLS
CONTRAST AGENTS
TARGETED PROBES
Dyes
Dye Labeling Kits
•
•
•
To enable intravital
imaging and
blood flow
imaging
Broad Applications
(2006)
•
•
Multiple NIR wavelengths:
–
AF: 647, 680, 750
–
Multiplexing
–
Spectral separation
Multiple chemistries:
–
Better specificity
–
Brighter
Patented Bodipy dye
portfolio
Quantum Dots
•
Expanded in-vivo utility
•
•
3 degrees of flexibility
Multiple NIR wavelengths:
–
AF: 647, 680, 750
–
NIR Nanocrystals
–
Multiplexing
–
Spectral separation
Scale (0.1mg, 1.0 mg)
Degree of Labeling Control
for brightness vs specificity
vs pharmacokinetics
Quantum Dots
•
Available Now
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Conjugation Kits
Available Now
Alexa Fluor NIR Dyes
•
•
Labelled colloidal agents
Labelled proteins
To enable
Customer Research in:
•
•
•
•
Cell Tracking (Now)
•
Quantum Dot
Qtracker® non-targeted solution
for tail vein injection
Apoptosis:
Angiogenesis:
Cancer:
Inflammation:
Qtracker® cell labeling kits
Confidential Custom
Services (Now)
•
Aimed at Pharma/Biotech
Available Now
Unparalleled flexibility
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Alexa Fluor NIR Dye (SAIVI) Protein Labeling Kits
•
Simple and easy to follow protocols
•
Reactive dye is pre-measured – no need to weigh out small quantities of dye
•
Procedure (including purification) takes ~2 hours, with little hands on time
•
Two kit sizes
Alexa Fluor® Protein Labeling Kit (1 mg)
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Alexa Fluor® Monoclonal Labeling Kit (100
ug)
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SAIVI TM Labeling Kits: Three degrees of flexibility
IgG
IgG
IgG
Effect of Modulating Agent on Degree of Labeling
DOL 6
NIR dye
DOL 3
NIR dye
DOL 1
NIR dye
Wavelength
A simple and quick protocol for variation and control of the
degree of labeling (DOL) allows for optimization of brightness
and specificity in in vivo applications.
Normalized Degree of Labeling
1.2
1
0.8
0.6
0.4
0.2
0
0 ul
750 nm
30ul
10 ul
Microliters of Added Modulating Agent
680 nm
647 nm
0.1 mg
Scale
1.0 mg
Control of Degree of Labeling (DOL) of a typical IgG
anitbody with Alexa Fluor 680 NIR dye using a SAIVI™
Alexa Fluor 680 0.1 mg Antibody/Protein Labeling Kit.
Alteration of DOL was achieved without a significant
change in protein volume or concentration. Final yield of
purified protein remains constant across the DOL range.
Three degrees of flexibility provide
unprecedented convenience.
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In-vivo imaging across the entire continuum
Molecular Targeting
Non-targeted
Intravascular
Cell Surface
Interstitial
Quantum Dots
Intracellular
Metabolic
Quantum Dots
Alexa Fluors
Alexa Fluors
Cellular Tracking
Passive
Proliferation
Uptake
Metabolism
Reporter Systems
Quantum Dots
Alexa Fluors
Post-mortem analysis
Physiology
Molecular
Quantum Dots
Alexa Fluors
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Alexa Fluor Dyes Simply The Best
• Brightness
• Photostability
• Color Selection
• pH Insensitivity
• Water Solubility
• Ease of Use
• Wide Selection
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•Broad Selection
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Immuno Histochemistry Workflow
Workflow Problems
Culture Cells
Fix cells
Permeabilise
Blocking Buffer
Primary Antibody
Secondary Antibody
Nuclear Stain
Low Signal Intensity
Photobleaching
Non-specific Binding
Mount
Microscopy
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Filter Quality
57
Semrock BrightLine® filters
• 2 to 4 times the brightness
• Twice the contrast
• Extremely low crosstalk
•
Unparalleled performance
– brightest & most discriminating filters for the fastest measurements
•
Unique spectral capabilities
– unlock new capabilities that you once only dreamed about!
•
Proven reliability for permanent performance
– work in hot, humid or corrosive environments
•
Highest batch-to-batch reproducibility
– ensure the repeatable manufacturing of your product
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Independent images taken by experts in the field
• Differences in film density and resulting overall film quality are readily apparent
Semrock Filters
Ion-Beam Sputtered
Hard Coating
E-beam Evaporated
Soft Coating
Major Competitor
Identical conditions
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BrightLine® spectra versus leading competitor
Exciter / emitter pairs for FITC
Competitor’s multiband set
Photographs of a Molecular Probes FluoCells #2
slide taken on an Olympus BX41 microscope using
a Spot Insight Color camera by Diagnostic
Instruments Inc. with competing DAPI/FITC/Texas
Red multiband filters sets
BrightLineTM multiband set
TM Multiband – visibly superior …
BrightLine
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… and, quantitatively superior!
Competitor
BrightLineTM
Color images were
captured by a Spot
Insight color CCD
camera by Diagnostic
Instruments Inc.
Analysis based on
monochrome images
captured by a
QImaging Retiga
cooled 12-bit CCD
camera
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Compared to the competitors’ best filter set …
Competitor
Semrock BrightLine®
Competitor
Semrock BrightLine®
Comparisons done under identical imaging conditions using an Olympus BX61WI microscope outfitted DSU spinning-disk confocal unit and a
Hamamatsu ORCA-ER monochrome CCD camera. RMK sample courtesy of Mike Davidson, Molecular Expressions™.
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Semrock for the ultimate in…
The Semrock BrightLine® family
When you want the best images !
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Brightness, Performance, Reliability !
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www.invitrogen.com/iProtocol
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www.invitrogen.com/iPath
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www.invitrogen.com/Antibodies
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Literature and brochures
Easy downloads from the web, or available on request.
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We wrote the book on fluorescence detection . . .
You can have your own copy!
www.probes.invitrogen.com
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Questions…
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