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Colorada – Fluorescence Labelsstarting at page222
Laguna Colorada (Bolivia)Image: Wikipedia
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Colorada – Fluorescence Labels
wavelength [nm]
B: Emission
A: Absorption Spectra of Colorada Fluorescent Dyes:
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NN N
N
N+
B-F
F
O
OH
MeOo BDP derivativeDipyrrometheneboron difluoride
N+ N
N
-O3S
O
ON
O
Oo Cyanine dye
ON O
NH
O
OH
O
o Coumarine dye
N
O
CNHN
OO OH
O
o Acenaphthopyrrole dye
Colorada – Fluorescence Labels
• based on 4 chemical classes:
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Colorada – Fluorescence Labels
Schematic presentation of the product‘s variations:
• different charges: o anionic
o cationic
o zwitterionic
o neutral
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Colorada – Fluorescence Labels
• different reactive groups:
o acid (carboxylic acid, COOH), diacid
o azide (N3)
o active ester (NHS-ester, COOX)
o iodoacetamide
o 2-pyridyldisulfideamine (PDA)
o aliphatic amine (NH2)
o dichlorotriazine (DCT)
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Colorada Dye Selection TableColorada Excitation
λmax [nm]Laser Excitation
SourceEmissionλmax [nm]
Stoke’sshift [nm] Reactive Groups Quencher Chemical
Class Alternative Dyes
425 N 425 Diode V 476 51 COOH, COOX, NH2, IOD QS35 COUM PF415
500 N 496 Ar, Diode B 506 10 COOH, COOX, NH2, N3, PDA QSY7 BDP FITC, FAM, BODIPY FL, AF488, PF488, OG488, PF505, OG514
515 N 515 Ar, Diode B 539 24 COOH, COOX, NH2, N3, PDA QSY7 BDP AF514, 6-JOE, 6-TET, PF520LSS530 N 529 Nd:YAG; Diode G 561 32 COOH, COOX, NH2, N3, PDA QSY9 BDP BODIPY R6G, AF532
550 C 550 Nd:YAG; Diode G; HeNe G 567 17 COOH, COOX KIT, NH2, IOD, PDA C5Q CYAN TAMRA, BODIPY TMR, CY3, DL549, PF546, AF546, AF555, PF555
550 Z 550 Nd:YAG; Diode G; HeNe G 566 16 COOH, COOX KIT, NH2, N3, IOD, PDA C5Q CYAN TAMRA, BODIPY TMR, CY3, DL549, PF546, AF546, AF555, PF555
550 A 553 Nd:YAG; Diode G; HeNe G 568 15 COOH, COOX KIT, NH2, IOD, PDA C5Q CYAN TAMRA, BODIPY TMR, CY3, DL549, PF546, AF546, AF555, PF555
555 N 551 HeNe G 572 12 COOH, COOX, NH2, N3, PDA C5Q BDP Rhodamine R, Cy3B570 N 573 Kr1 612 39 COOH, COOX, NH2, N3, PDA C5Q BDP AF568577 N 577 Kr1 592 22 COOH, COOX, NH2, N3, PDA QS21 ACEN Cy3.5, PF590580 N 580 Kr1 592 22 COOH, COOX, NH2, N3, PDA QS21 BDP Cy3.5, PF590
600 N 593 HeNe Y; Diode Y 624 37 COOH, COOX, N3, PDA QS21 BDP TEXAS RED, BODIPY TR, AF594, AF610, PF 610
630 N 629 HeNe R, Diode R1 646 17 COOH, COOX, N3, PDA QSY21 BDP BODIPY 630/650, AF633, PF633, EB 10/30
645 C 641 HeNe R; Diode R1 663 22 COOH, COOX (& KIT), NH2, IOD, PDA QSY21 CYAN Cy5, BODIPY 650/665, DL649, AF 647, PF647
645 Z 643 HeNe R; Diode R1 665 22 COOH, COOX KIT, NH2, IOD, PDA QSY21 CYAN Cy5, BODIPY 650/665, DL649, AF 647, PF647
645 A 648 Kr2, HeNe R; DiodeR1-R2 667 21 COOH, COOX (& KIT), NH2, N3, IOD, PDA QSY21 CYAN Cy5, BODIPY 650/665, DL649, AF 647, PF647
678 C 679 Kr2, Diode R2-R3 707 28 COOH, COOX (KIT), PDA QSY21 CYAN Cy5.5, AF660, AF680, PF680678 Z 678 Kr2, Diode R2-R3 705 27 COOH, COOX KIT, NH2, N3 QSY21 CYAN Cy5.5, AF660, AF680, PF680678 A 677 Kr2, Diode R2-R3 703 26 COOH, COOX KIT QSY21 CYAN Cy5.5, AF660, AF680, PF680770 C 768 Diode NIR1 790 22 COOH, COOX , DCT, NH2, CYAN PF780
770 A 772 Diode NIR1 807 29 COOH, COOX, DCT, NH2 CYAN PF780
800 C 802 Diode NIR2 840 38 COOH, COOX , DCT, NH2 CYAN
800 A 802 Diode NIR2 847 45 COOH, COOX , DCT, NH2 CYAN
830 C 827 Diode NIR3 867 40 COOH, COOX, DCT, NH2 CYAN
830 A 826 Diode NIR3 851 25 COOH, COOX, DCT, NH2 CYAN
Cy5, Alexa Fluor 647(Invitrogen) Cy5, Alexa Fluor 647(Invitrogen) Cy5, Alexa Fluor 647(Invitrogen)
TAMRA(Invitrogen) TAMRA(Invitrogen) TAMRA(Invitrogen)
Cy5.5, Alexa Fluor 680(Invitrogen) Cy5.5, Alexa Fluor 680(Invitrogen) Cy5.5, Alexa Fluor 680
(Invitrogen)
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• Literature
Colorada – Fluorescence Labels
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Literature request AppliChem Request Literature Brochures •AppliCation Notes • Posters
Product Code Description
Applications 4 pages of background information
A55 AppliCations No. 1 DNA-ExitusPlus
A91 AppliCations No. 2 Biological Buffers
A84,E AppliCations No. 4 AppliCoat Plate Stabilizer
A95,E Applications No. 6 Gel Filtration
A96,E AppliCations No. 7 SeparateIT
A102,E Applications No. 8 Colorada
A103,E Applications No. 9 CheLuminate
A107,E Applications No. 10 SafetyFirst Caps
A114,E Applications No. 12 Cell Proliferation Assay XTT
A115,E Applications No. 13 Cytokines & Growth Factors
A118,E AppliCations No. 14 Protease Inhibitors
A131,E AppliCations No. 15 AppliFect Transfection Reagents
Brochures comprehensive information
A27 Biological Buffers
A53 Detergents
A26,E Gel Electrophoresis Size Marker
A71,E Immunoassay Buffer
A72 Contamination by Nucleic Acids
A24 Safety First: Banish Mycoplasma
A25 Transfer Membranes
Flyer/Posters condensed & clear
A119,E Flyer Support&More No.1 Mycoplasma in Cell Cultures
A139,E Flyer Support&More No. 9 Colorada Labeling Kits
A35 Periodic Table of Elements (folded)
A132,FOLD GHS Classification (Engl. & Ferman, folded)
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• Applications
o Peptide labeling
Colorada – Fluorescence Labels
o Amino acid labeling
o Protein labeling / Antibody labeling
o RNA and DNA labeling
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Colorada – Fluorescence Labels
Merge between a normal plasma labeled with Colorada 645 Z – Iodoacetamide (C5NIASO3) and plasma from a FSGS patient (glomerulosclerosis) labeled with Colorada 550 Z – Iodoacetamide (C3NIASO3).
• Example: DIGE (Differential Display Gel Electrophoresis),
2-D gel of labeled plasma proteins.
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Colorada – Labeling Kits
Step 1Dye Solution Preparation
Step 2 Antibody Solution Preparation
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Colorada – Labeling Kits
Step 3Conjugation Procedure
Step 4Isolation of the Conjugate
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Colorada – Labeling Kits
Product No.*
Chromophore Abs. λ max. Em. λ max. Molar extinction coefficient
A9368,0001A9368,0005
Colorada 550 553 nm 568 nm 150000 M-1cm-1
A9404,0001A9404,0005
Colorada 645 648 nm 667 nm 250000 M-1cm-1
A9517,0001A9517,0005
Colorada 678 677 nm 703 nm 195000 M-1cm-1
Colorada Antibody Labeling Kits
*1 or 5 labeling reactions of 1 mg protein each
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Colorada – Labeling Kits
Product No.*
Chromophore Abs. λ max. Em. λ max. Molar extinction coefficient
A9368,0001A9368,0005
Colorada 550 553 nm 568 nm 150000 M-1cm-1
A9404,0001A9404,0005
Colorada 645 648 nm 667 nm 250000 M-1cm-1
A9517,0001A9517,0005
Colorada 678 677 nm 703 nm 195000 M-1cm-1
Product No.**
Chromophore Abs. λ max. Em. λ max. Cyanin structure
Molar extinction coefficient
A9426,0001A9426,0005
Colorada 490 DIGE
484 nm 498 nm Cy2 110000 M-1cm-1
A9447,0001A9447,0005
Colorada 550 DIGE
548 nm 567 nm Cy3 150000 M-1cm-1
A9498,0001A9498,0005
Colorada 645 DIGE
641 nm 663 nm Cy5 250000 M-1cm-1
Colorada Antibody Labeling Kits
Colorada DIGE Protein Labeling Kits
*10 or 50 labeling reactions
*1 or 5 labeling reactions of 1 mg protein each
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• Literaturesoon available
Colorada – Labeling Kits
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Phosphate buffered saline (PBS)
1. powder• PBS buffer (10X Dulbecco's) - Powder A0965 • PBS buffer (1X Dulbecco's) - Powder A1908 • PBS buffer (1X Dulbecco's) - Powder A0964
2. solution• PBS / Tween® - Solution A3814
Blocking solution, e.g. in ELISA
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• pH 7,4 for final volumes of: 100 ml, 200 ml, 500 ml, and 1 L
• pH 7,2 for a final volume of: 1 L
Phosphate buffered saline (PBS)
On popular demand: new at AppliChem
3. PBS Buffer tablets
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TransfectionGFP Drosophilaimage: Univ of California, San Diego website
AppliFect – Transfection Reagentsstarting at page 114
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Transfection Reagents
nucleus
lipoplex
cell membrane
endosome
DNA entry during Mitosis
cytosol
DNA
cationic lipid
co-lipids
release of DNA
endocytosis
!
!
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Transfection Reagents
AppliFect – fast and easy protocol
1. seed cells
2. prepare DNA-solution
+ AppliFect
3. complex
4. incubate 5. reporter gen assay
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Liposomes consisting of polycationic transfection reagent + a neutral co-lipid
AppliFect - for routine transfection of plasmid DNA into eukaryotic cells
o easy - ready-to-use reagent
o effective - highest expression rates
o robust - serum doesn‘t interfere with transfection
Transfection Reagents
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• 1. AppliFect
Transfection ReagentsAppliFect example: COS-7 cells GFP transfected
transmitted light
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o Lipofectamine (Invitrogen) 672,00 €/1 ml [German list price]o FuGENE HD (Promega) 369,00 €/1 ml [German list price]o GeneJuice (Novagen) 306,00 €/1 ml [German list price]o Metafectene (Biontex)o Unifectin (Novagen)o Ribojuice (Novagen)o Nanofectin (Novagen)
• Competitors
Transfection Reagents
• AppliFect - key benefits:o High efficiencyo Competitive Price
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o Lipofectamine (Invitrogen) 672,00 €/1 ml [German list price]o FuGENE HD (Promega) 369,00 €/1 ml [German list price]o GeneJuice (Novagen) 306,00 €/1 ml [German list price]o Metafectene (Biontex)o Unifectin (Novagen)o Ribojuice (Novagen)o Nanofectin (Novagen)
• Competitors
Transfection Reagents
• AppliFect - key benefits:o High efficiencyo Competitive Price 188,40 €/1 ml [German list price]
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• 4. AppliFect SI
Transfection Reagents
• 3. AppliFect Insect - DNA for insect cells
• 2. AppliFect LowTox - pDNA, for sensitive cells
232,00 €/1 ml [German list price]
• 1. AppliFect - (plasmid) pDNA, RNA
- for siRNA, miRNA, RNA
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Transfection Reagents
Promote AppliFect
o literature: application notes comprehensive product information
o promotions
o sampling