FluoProbes® FT-FP488_ FluoProbes®488 labeling agents

Product Information

A great for labeling biomolecules with fluorescent green emission (alternative to FITC)

Product name MW exc\em. max. mol. abs. Quantum cat.number (g·mol-1) (nm) (M-1cm-1) yield . M+ (%) Fluoprobes®488 - Carboxyl group 804 590 (L) FP-BA6790, 1mg 493 / 519 80 000 82 Fluoprobes®488 - NHS 981 687 τfl = 3.3 ns (K) FP-BA6800, 1mg ® Fluoprobes 488 - Maleimide CF260(ε260/εmax)= 0.2 1067 712 (M) FP-BA6810, 1mg CF260(ε260/εmax)= 0.1 Fluoprobes®488 - IodoAcetamide  A superior alternative to (L) FP-FH9780, 1mg , Cy2, A488... Fluoprobes®488 - Hydrazide  Bright green (L) FP-B38820, 1mg Fluoprobes®488 - Azide 903 M+: Mass without counter-ion (L) FP-YE4970, 1mg Fluoprobes®488 - Alkyne 740 (L) FP-LV4430, 1mg Fluoprobes®488 Protein Lab.Kit - (V) FP-BE3750, 1 kit

Storage: (L): at +4°C (K) : at +4°C (long term at –20°C) (M): at –20°C

Fluoprobes®488 is the most popular green emitter fluorescent label, part our the Fluoprobes® dyes series, alternative to FITC, Cy2, A488.

Scientific and technical Information - Label

FluoProbes®488 is a very bright and photostable green dye*

Features:  can be excited by any source used for fluoresceins, i.e. the 488 line of Argon .  compatible with standard filters for Fluoresceins

 Bright green fluorescence (λexc./λem.: 593/519nm): FP®488 shows elevated extinction coefficient (ε at λmax.: 95 000 M-1cm-1 ) and quantum yield (QY>80%). see comparison [c]  Low background compared e.g. with fluoresceins: see comparison [c], and note [b] Absorption and emission spectra  Unrivaled photostability at physiological pH-values and upon light exposure (e.g. during scanning This label is available as several derivatives procedures, storage)., hence minimal fading and (see above) and already coupled to several ultimate shelf-life... See note [a] specific ligands (see related products). It is  pH-independent fluorescence between pH 4 and 10 recommended for labeling at high coupling ratios.  Lowest self-quenching, even at high ratios: see note [b]

 NHS-ester and maleimide derivates show excellent solubility in polar solvents like DMF, DMSO or [c]:See the comparisons with competitors acetonitrile. [email protected] FluoProbes®, powered by P.1 [email protected] [email protected] Contact your local distributor FluoProbes® FT-FP488_ [b] FP®488 fair hydrophilicity (when coupled) allow As a result, FluoProbes®488 is: for lower background than other dyes, i.e. , - a superior alternative to FITC, Cy2, A488 and limits occurring of self-quenching, even at high - suits most fluorescent techniques, ratios. including FCM, microplate assays, microarrays, microscopy, providing excellent sensitivity with low background. [a] FP®488 elicits the better photostability amongst all tested green dyes, including references FITC, Cy2, A488. Consequently, longer integration of signal in digital imaging can be achieved to gain detection of low abundance molecules, signal amplification method may be avoided, re- analysis of samples remain as quantitative you do not need to use (additional price and time) antifading agents or their additives with correlated cytotoxicity. It also confers advantage regarding shelf-life, e.g. in for manufacturing diagnostics.

+

Applications: FP488 performs especially advantageously in (CFSM), thanks its unrivaled photostability compatible with required long acquisition time (scanning). It out-performs as well in any application where light exposure or stability is critical, for example for manufacturing diagnostics. To that regard, FP488 is superior to all other tested green dyes. FP488 suits Wide Field Microscopy (WFM), (FCM), MicroArrays thanks its high brightness. It replaces advantageously standard green dyes as FITC, Cy2. FP488 suits fluorescence polarization analysis and Life Time resolving fluorescence. Life time is 3.2-5ns. It distinguish from most other green dyes (FITC) by its negative anisotropy. FP488 can be successfully used by Localization microscopy SPDM / SPDMphymod technologyw. FP488 can be used as a iron indicator.

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Scientific and technical Information - derivates

Fluoprobes®488 is available as different derivatives, suiting standard chemistry methods. MW Added Mass FP488 carries a negative charge, that is Fluoprobes®488 - Carboxyl group 804 588 transferred after conjugation. FP-BS5610 Fluoprobes®488 - NHS 981 573 The MW of conjugate can be calculated FP-BS5620 with following table (MW without counter Fluoprobes®488 - Maleimide ion (M+:), less weight of the removed group FP-BS5630 1067 711 during the coupling procedure):

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Carboxylic derivatives are can be used for any kind of spectroscopy, and coupled to biomolecules by conventional chemistry, i.e. after activation at the carboxy group by carbodiimides (EDAC). Amine derivatives can be used for any kind of spectroscopy, and coupled to biomolecules by conventional chemistry, i.e. by reaction with aldehydes, or with carboxyls by amidation mediated by carbodiimides (EDAC). Please refer to the literature for protocols, or the technical sheet FT-52005A (EDAC): Storage : Carboxylic acid and mine derivatives are stored at ambient temperature and is stable for at least three years.(L)

NHS-ester derivatives are suited for direct labeling of amino groups in proteins and aminated DNA/RNA. The chemical group N-hydroxysuccinimidyl (NHS) reacts specifically with primary (–NH2) and secondary amines (-NH-) (in fact on its deprotonated form) in aqueous phase or at pH 8 (compatible with pH7 to 10) in PBS buffer (other buffer devoid of amines are possible) at a ratio of 1-6 over amine content. I.e. amines present in proteins (Lys aminoacid) and in a lower proportion on NH2 located in terminal peptidic chains. The reaction competes with hydrolysis that increases with pH, and with the high dilutions of the molecule that should be labeled. Please refer to the literature for protocols, or the technical sheet FT-BA680 (NHS-FluoProbes labels): Storage : NHS-esters can be stored at 0-4°C, stable for several months. They should be protected from moisture and light.(K)

Maleimide derivatives are suited for labeling of thiol groups, e.g. specific labeling of cysteine of proteins or peptides. The maleimide group reacts very specifically with sulfhydryls –SH at neutral pH 6.5-7. The reaction is rapid (a few minutes for cysteine), but in the absence of –SH, maleimide stay well stable. In usual conditions, one should start with a ratio of 10-20 moles of maleimide per mole of protein. Please refer to the literature for protocols, or the technical sheet FT-BA681 (Maleimide-FluoProbes labels): Storage : Maleimide derivatives can be stored at 0-4°C, stable for several months, or at –20°C for long term. They should be protected from moisture and light.(M)

Hydrazide derivatives give a variety of reactions: .react specifically with aldehydes and ketones, forming an hydrazine bond. .react with carboxyls , that better occur with activation by a carbodiimide (EDC). .react also with amines forming an intermediate Shiff's base that can be further stabilized by reduction . .other reactions are possible as well (with cytosine, NHS) Hydrazide provides thus privileged methods to conjugate a variety of biomolecules: glycoproteins, glycolipids, sialic acids and sugars, steroids, LDL and nucleic acids, but also N-terminal serine and threonine residues in proteins. For reducing sugars (containing free CHO groups), direct conjugation is possible, but most other applications require a reducing or an oxidising step to generate CHO groups from carboxyls or from cis-diols. Please refer to the literature for protocols, or the technical sheet FT-CE0640 (Hydrazide-FluoProbes labels): Storage : Hydrazide derivatives should be stored at 0-4°C and are stable for at least one year.

Azide derivatives yield useful reactions: .they are also popular for their participation in bioorthogonal reactions, the “Click Chemistry” (with to alkyne-modified molecules, forming a stable triazole bond), and the Staudinger ligation (with Phosphine-modified molecules, forming an amide bond). .they are commonly used as a way to introduce an amine group, .they participate to other kind of reactions as well, for example azide react with carbonyls or ketones to form amine or amides (Schmidt reaction); and arylazides react with amines, freeH). Please refer to the literature for protocols, or the technical sheet FT-YE4970 (Azide-FluoProbes labels) and FT-ZL5530 (Click Chemistry reagents):

Storage : Azide derivatives should be stored at 0-4°C and are stable for at least one year. (L)

Alkyne derivatives provide a nice partner for Click Chemistry based conjugations with Azides: the Alkyne-FluoProbes can be click-conjugated to azide-modified molecules, through a stable triazole bond. Please refer to the literature for protocols, and above information for Azide protocols. Storage : Alkyne derivatives should be stored at 0-4°C and are stable for at least one year. (L)

General advice In any bioconjugation, a calibration of dye/biomolecule ratio may be needed to optimize the labeling level depending on molecule and application, i.e. adjust concentration weight of the FluoProbes® dye / weight of protein or peptide. Then the parameters of the detection instrument should also be sat properly for FluoProbes dye (see above/label).

[email protected] FluoProbes®, powered by P.3 [email protected] [email protected] Contact your local distributor FluoProbes® FT-FP488_ References for FluoProbes488 labeled products:

Brunner Yannick et al; Molecular & Cellular Proteomics 6.6 1007-1017; "Proteomics Analysis of Insulin Secretory Granules", Article [FP546nm anti-rabbit, FP546nm anti-mouse, FP488nm anti-rabbit, FP488nm anti-mouse, FP488nm anti-pig used in LSCM LSM 510 Meta, Zeiss] Segain Jean-Pierre ; The Journal of Immunology, 2007, 179: 901-909; "Urotensin II is a New Chemotactic Factor for UT Receptor-Expressing Monocytes"; Abstract; [anti-rabbit FluoProbes 488 Ab] Norez Caroline et al; JPET 325:89-99, 2008; "Proteasome-Dependent Pharmacological Rescue of Cystic Fibrosis Transmembrane Conductance Regulator Revealed by Mutation of Glycine 622"; Abstract [FluoProbes 488 secondary Ab] Galiana Eric et al; Environmental Microbiology, Volume 10 Issue 8, Pages 2164 – 2171 (2008) ; "Phytophthora parasitica biofilm formation: installation and organization of microcolonies on the surface of a host plant"; Abstract [FP488 Rabit IgG Ab] Zanella Sébastien et al; The Journal of Neuroscience, February 13, 2008, 28(7):1745-1755; "Necdin Plays a Role in the Serotonergic Modulation of the Mouse Respiratory Network: Implication for Prader-Willi Syndrome"; Abstract [FluoProbes 488 Donkey anti Mouse and Pig IgG Ab] Vandebrouck A. et al; The FASEB Journal. 2007;21:608-617 ; "Regulation of capacitative calcium entries by α1-syntrophin: association of TRPC1 with dystrophin complex and the PDZ domain of α1-syntrophin"; Abstract; Article; [FluoProbes 488 Donkey anti Mouse IgG Ab; Fura2- AM] Grozio Alessia at al; International Journal of Cancer Volume 122 Issue 8, Pages 1911 – 1915; "Natural agents targeting the 7-nicotinic-receptor in NSCLC: A promising prospective in anti-cancer drug development"; Abstract [NHS-FP488] Dhumpa Raghuram ; Master Thesis Stockholm Sweden 2007; "Development Of A Microfluidic System For Bead Based Microarray Application"; Article;[Rabit IgG-FluoProbes488, read with ZeptoREADER CDD camera (exc.532&635nm) and CLSL LSM 510 (Zeiss) exc.Argon, HeNe 633, and HeNe 561) Kuczewski Nicola ; The Journal of Neuroscience, July 2, 2008, 28(27):7013-7023; "Backpropagating Action Potentials Trigger Dendritic Release of BDNF during Spontaneous Network Activity"; Abstract [FluoProbes647]

Other FluoProbes488 labeled products: *Other FluoProbes488 conjugates: FluoProbes488 labeled Anti IgG antibodies FP-HO1220: FP488-Cysteine FP-BA2221: Streptavidin-FP488 FP-BC6381: Avidin-FP488 FP-BA6820: Biotin-FP488 FP-BH9390: AnnexinV-FP488/FCM FP-BH4140: AnnexinV-FP488/Conf.Microscopy FP-BY6820:AnnexinV for in-vivo FP-YE5180: Phalloidin-FP488 FP-SM5650: BSA-FP488 FP-IL7530: Aminoallyl-UTP-FP488 FP-IM1110: Aminoallyl-dUTP-FP488 FP-IM4600: dCTP-FP488 FP-KA6580: CTP-FP488 Other FluoProbes488 – labeled products : please inquire

RFU Other FluoProbes labels: 1,0 Fluoprobes® provides a full range of to covers any applications, spanning from 390nm to 800nm. Fluoprobes® dyes are designed for labeling biomolecules in advanced fluorescent detection 0,8 techniques. Applications include multiple labeling, FRET, Quenching, polarisation anisotropy fluorescence, FP®390 - Em. 0,6 FP®488 - Em. FP®532 - Em. and life time resolved fluorescence, with protein as well as with nucleic acids, as well as dying materials. FP®547H - Em. FP®565 - Em. FP®594 - Em. FP®647H - Em. Please see a presentation of selected most popular and remarkable FluoProbes labels in 0,4 FP®682 - Em. FP®782 - Em. standard applications (i.e. blue, green, orange, red, infrared), or at pages B51-B57 of the BioSciences catalogue, and updated list of FluoProbes dyes NHS esters at 0,2

nm http://www.interchim.fr/ft/F/FPlistN.pdf . 0,0 350 450 550 650 750 850

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Ordering information

Catalog size quantities and prices may be found at www.interchim.com/interchim/customers/ Please inquire for higher quantities (availability, shipment conditions).

For any information, please ask : FluoProbes® / Interchim; Hotline : +33(0)4 70 03 73 06

Disclaimer : Materials from FluoProbes® are sold for research use only. Please consult FluoProbes for other uses. FluoProbes® is not liable for any damage resulting from handling or contact with this product. Rev. R10E-J06E-H04E/01

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