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ANTIBODIES CONJUGATED TO ATTO DYES For Flow Cytometry & Immunofluorescence

Advantages of ATTO Dyes

Increased Photostability

Outstanding Brightness

High Thermal Stability

Intense Signals (εmax > 90,000)

Resistant to Environmental Changes

Insignificant Isomerization

ATTO 488Alternative to Alexa Fluor® 488

Outperforms FITC, R-PE, and Cy™2

ATTO 590Alternative to Texas Red® and Alexa Fluor® 594

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Color ATTO Dye MW λabs λem εmax Isomers [g/mol] [n/m] [n/m] [l/(mol*cm)]

Green ATTO 488 590 501 523 90,000 No significant isomerization

Red ATTO 590 592 594 624 120,000 No significant isomerizaiton

Properties

Brilliant Dyes – Superior Results !

In-house antibody labelling with the next generation of dyes ensures consistent quality. Our application department provides full technical support.

Photostability of ATTO 488

Photostability of ATTO488 compared to com-mon dyes in water.Excitation at 488 nmwith a 1 W Argon-Ionlaser.

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Spectra of ATTO 488 and ATTO 590 dyes

Panel of ATTO-conjugated Antibodies see Page 3

L A B E L L E D A N T I B O D I E S

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Test Results ATTO 488

ATTO 488, a superior label for Flow Cytometry analysis

FIGURE: BJAB cells were stained with MAb to BAFF-R (human) (11C1) (Prod. No. ALX-804-807) andrevealed with PAb to Mouse lgG1 labelled with FITC, Alexa Fluor® 488 or with ATTO 488 (Prod. No. ALX-211-204TD).

ATTO 488 exhibits a brighter fluorescence due to strongabsorbance and high quantum yield

FIGURE: Comparison of the fluorescence index between MAb to TLR4 (human) (HTA125) (Prod. No. ALX-804-419) conjugated to FITC (Prod. No. ALX-804-419F) or ATTO 488 (Prod. No. ALX-804-419TD).

2000400060008000

100001200014000160001800020000

8.00 4.00 2.00 1.00 0.50 0.25 0.13 0.06 0.03 0.02 0.01

µg /ml

MAbAA to TLR4 FITC

MAbAA to TLR4 ATTO 488

ATTO 590, a brighter alternative to Texas Red®

FIGURE: HeLa cells are stained with MAb to BAP31 (CC-1) (ALX-804-812) and revealed with PAb to RatIgG (ATTO 590) (ALX-211-059TM) or PAb to Rat IgG (Texas Red®).

Fluorescence of ATTO 488 compared to Alexa Fluor® 488

FIGURE: HeLa cells are stained with PAb to Fis1 (Prod. No. ALX-210-907) and revealed with PAb to Rabbit IgG (ATTO 488) (Prod. No. ALX-211-650TD) or PAb to Rabbit IgG Alexa 488.

ATTO 488 Alexa Fluor® 488

ATTO 488 with its outstanding brightness outperforms R-PE

FIGURE: PAb to Rat IgG (ATTO488)(Prod. No. ALX-211-059TD) is more efficient than PAb to Rat IgG (R-PE) (Prod. No. ALX-211-053) for the detection of endogenoushuman BCMA with MAb to BCMA (human) (Vicky-1) (Prod. No. ALX-804-151) in U266 cells.

Test Results ATTO 590

ATTO 590 Texas Red®TrademarksTexas Red® and Alexa Fluor® are registered trademarks of Molecular Probes, Inc.

Cy™ is a trademark of GE Healthcare (formerly Amersham Biosciences).

FIGURE: HeLa cells are stained with MAb to Tubulin and revealed with PAb to Mouse IgG (ATTO 488)(Prod. No. ALX-211-205TD). Pictures taken at time 0 min (left picture) or 1 min (right picture) do not showany noticeable photo bleaching.

Photostability of ATTO 488

L A B E L L E D A N T I B O D I E S

Purified (PF) = Purified (Preservative free); FC = Flow Cytometry; ICC = Immunocytochemistry; IP = Immunoprecipitation; IHC = Immunohistochemistry (FS = Frozen Sections, PS = Paraffin Sections); WB = Western blot

ATTO Dye Labelled Antibodies

Apoptosis

MAb to BAFF (human) (Buffy-1) (ATTO488)ALX-804-128TD-T100 100 tests CLONE: Buffy-1. ISOT YPE: Rat IgG2a. IMMUNOGEN: Recom-binant human soluble BAFF (aa 83-285). SPECIFICIT Y: Recog-nizes membrane-bound human BAFF. Detects endogenous pro-tein by FC. APPLICATION: FC, ICC.

MAb to Perforin (mouse) (CB5.4) (ATTO 488)ALX-804-057TD-T100 100 tests CLONE: CB5.4. ISOT YPE: Rat IgG2a. IMMUNOGEN: Recombinantmouse perforin (aa 98-534). SPECIFICIT Y: Recognizes mouseperforin (region aa 402-534). APPLICATION: FC, ICC.

Inflammation

MAb to TLR2 (human) (TL2.1) (ATTO 488)ALX-804-323TD-T100 100 tests CLONE: TL2.1. ISOT YPE: Mouse IgG2a. IMMUNOGEN: CHO cellsstably transfected with human TLR2. SPECIFICIT Y: Recognizeshuman TLR2. Does not cross-react with human TLR1 or TLR4.APPLICATION: FC.

MAb to TLR4 (human) (HTA125) (ATTO 488)ALX-804-419TD-T100 100 tests CLONE: HTA125. ISOT YPE: Mouse IgG2a. IMMUNOGEN: Ba/F3 cellline expressing human TLR4. SPECIFICIT Y: Recognizes humanTLR4 cell surface antigen. APPLICATION: FC.

MAb to TLR4/MD-2 (mouse) (MTS510)(ATTO 488)ALX-804-430TD-T100 100 tests CLONE: MTS510. ISOT YPE: Rat IgG2a. IMMUNOGEN: Ba/F3 cellsexpressing mouse TLR4 and MD-2 complex. SPECIFICIT Y: Re-cognizes the mouse TLR4/MD-2 complex. APPLICATION: FC.

Secondary Antibodies

PAb to Mouse IgG1 (ATTO 488)ALX-211-204TD-C100 100 µg From goat. IMMUNOGEN: Mouse IgG1. SPECIFICIT Y: Recognizesthe heavy chain of mouse IgG1. Minimally cross-reacts with hu-man immunoglobulins. APPLICATION: FC, ICC.

PAb to Mouse IgG1 (ATTO 590)ALX-211-204TM-C100 100 µg From goat. IMMUNOGEN: Mouse IgG1. SPECIFICIT Y: Recognizesthe heavy chain of mouse IgG1. Minimally cross-reacts with hu-man immunoglobulins. APPLICATION: ICC.

PAb to Rat IgG (ATTO 488)ALX-211-059TD-C100 100 µgFrom goat. IMMUNOGEN: Rat IgG. SPECIFICIT Y: Recognizes the heavy and light chain of rat IgG, as well as the light chain of rat IgM and IgA. Minimally cross-reacts with mouse immunoglobu-lins. APPLICATION: FC, ICC.

PAb to Rat IgG (ATTO 590)ALX-211-059TM-C100 100 µgFrom goat. IMMUNOGEN: Rat IgG. SPECIFICIT Y: Recognizes the heavy and light chain of rat IgG, as well as the light chain of rat IgM and IgA. Minimally cross-reacts with mouse immunoglobu-lins. APPLICATION: ICC.

PAb to Mouse IgG (ATTO 488)ALX-211-205TD-C100 100 µgFrom goat. IMMUNOGEN: Mouse IgG. SPECIFICIT Y: Recognizes the heavy chain of mouse IgG1, IgG2a, IgG2b and IgG3. Mini-mally cross-reacts with human immunoglobulins. APPLICATION: FC, ICC.

PAb to Mouse IgG (ATTO 590)ALX-211-205TM-C100 100 µg From goat. IMMUNOGEN: Mouse IgG. SPECIFICIT Y: Recognizesthe heavy chain of mouse IgG1, IgG2a, IgG2b and IgG3. Min-imally cross-reacts with human immunoglobulins. APPLIC ATION: ICC.

PAb to Rabbit IgG (ATTO 488)ALX-211-650TD-C100 100 µg From goat. IMMUNOGEN: Rabbit IgG. SPECIFICIT Y: Recognizesthe heavy and light chain of rabbit IgG, as well as the light chainof rabbit IgM and IgA. Minimally cross-reacts with human andmouse immunoglobulins. APPLICATION: ICC.

PAb to Rabbit IgG (ATTO 590)ALX-211-650TM-C100 100 µg From goat. IMMUNOGEN: Rabbit IgG. SPECIFICIT Y: Recognizesthe heavy and light chain of rabbit IgG, as well as the light chainof rabbit IgM and IgA. Minimally cross-reacts with human andmouse immunoglobulins. APPLICATION: ICC.

Lymphocyte Activation Gene-3 (LAG-3) Lymphocyte activation gene-3 (LAG-3; CD223) is a CD4 homolog known to be selectively expressed

in activated T and NK cells. LAG-3 negatively regu- lates T cell function and homeostasis. It was shown that LAG-3 surface expression can also be found on

w activated B cells and LAG-3 was proposed as a neww marker of T cell induced B cell activation (Kisielow

et al.; Eur. J. Immunol. 35, 2081 (2005)).AAs a soluble molecule, sLAG-3 activates antigen-pre-senting cells through MHC class II signalling, lead-ing to increased antigen-specifi c T cell responses invivo y . In addition to being a Th1 marker, sLAG-3 maywell be an important effector molecule regulating well be an important effector molecule regulating

the immune response to tumor antigens (Triebel,et al.; Cancer Lett. 2005 (in press)).

MAb to LAG-3 (human) (17B4) (ATTO 488)ALX-804-806TD-T100 100 testsCLONE: 17B4. ISOT YPE: Mouse IgG1. IMMUNOGEN: Synthetic peptide corresponding to 30 aa (GPPAAAPGHPLAPGPHPAAPSS-WGPRPRRY) from the fi rst N-terminal D1 domain of human LAG-3 (lymphocyte activation gene-3). SPECIFICIT Y: Recognizes the 30 aa extra-loop of the fi rst N-terminal D1 domain of human LAG-3. APPLICATION: FC, ICC.

MAb to LAG-3 (human) (17B4) (ATTO 590)ALX-804-806TM-T100 100 testsCLONE: 17B4. ISOT YPE: Mouse IgG1. IMMUNOGEN: Synthetic peptide corresponding to 30 aa (GPPAAAPGHPLAPGPHPAAPSS-WGPRPRRY) from the fi rst N-terminal D1 domain of human LAG-3 (lymphocyte activation gene-3). SPECIFICIT Y: Recognizes the 30 aa extra-loop of the fi rst N-terminal D1 domain of human LAG-3. APPLICATION: ICC.

Immunology

FIGURE: Detection of endogenous mouse TLR4 by FACS analysis using the MAb to TLR4/MD-2 (mouse) (MTS510) (ATTO 488) (Prod. No. ALX-804-430TD). METHOD: WEHI cells were stained with MAb to TLR4/MD-2 (mouse) (MTS510) labelled with FITC (Prod. No. ALX-804-430F) or with the new ATTO 488 dye. An isotype control labelled with FITC was used as reference.

–– MAb to TLR4/MD-2 (MTS510) (ATTO 488)

–– MAb to TLR4/MD-2 (MTS510) (FITC)

–– Rat IgG2a (FITC)

MAb to LAG-3 (human) (17B4) (ATTO 647)ALX-804-806TS-T100 100 tests CLONE: 17B4. ISOT YPE: Mouse IgG1. IMMUNOGEN: Synthetic peptide corresponding to 30 aa (GPPAAAPGHPLAPGPHPAAPSS-WGPRPRRY) from the fi rst N-terminal D1 domain of human LAG-3 (lymphocyte activation gene-3). SPECIFICIT Y: Recognizes the 30 aa extra-loop of the fi rst N-terminal D1 domain of human LAG-3. APPLICATION: FC, ICC.

Related Products

LAG-3 (human):Fc (human) (rec.)ALX-522-078-C050 50 µg Produced in CHO cells. The sequence coding for the 4 extra-cellular Ig-like domains of human LAG-3 (D1-D4) is fused to the Fc portion of human IgG1. BIOLOGICAL AC TIVIT Y: Inhibits binding of MAb to LAG-3 (human) to LAG-3. Induces matura-tion of human dendritic cells. APPLICATION: FC (binds MHC class II molecules).LIT: Expression and release of LAG-3-encoded protein by humanCD4+ T cells are associated with IFN-gamma production: F. An-nunziato, et al.; FASEB J. 10, 769 (1996) Preferential Th1 profile of T helper cell responses in X-linked (Bruton‘s) agammaglobuline-mia: A. Amedei, et al.; Eur. J. Immunol. 31, 1927 (2001) Activetuberculosis in Africa is associated with reduced Th1 and increasedTh2 activity in vivo: C. Lienhardt, et al.; Eur. J. Immunol. 32, 1605(2002) LAG-3 (CD223) reduces macrophage and dendritic celldifferentiation from monocyte precursors: S. Buisson & F. Triebel;Immunology 114, 369 (2005)

MAb to LAG-3 (human) (17B4)ALX-804-806-C100 100 µg ALX-804-806B-C100 Biotin 100 µg ALX-804-806F-C100 FITC 100 µg CLONE: 17B4. ISOT YPE: Mouse IgG1. IMMUNOGEN: Synthetic peptide corresponding to 30 aa (GPPAAAPGHPLAPGPHPAAPSS-WGPRPRRY) from the fi rst N-terminal D1 domain of human LAG-3 (lymphocyte activation gene-3). SPECIFICIT Y: Recognizes the 30 aa extra-loop of the fi rst N-terminal D1 domain of human LAG-3. APPLICATION: ELISA, FC, ICC, IHC (FS), IP, WB.

MAb to to LAG-3 (human) (11E3)ALX-804-805-C100 100 µg CLONE: 11E3. ISOT YPE: Mouse IgG1. IMMUNOGEN: Recom-binant human LAG-3 (lymphocyte activation gene-3). SPECIFICIT Y: Recognizes the fi rst N-terminal D1 domain of human and monkey LAG-3. APPLICATION: ELISA, ICC, IHC (FS), IP, WB.

LAG-3, Soluble (human) Detection SetAPO-54N-017-KI01 1 Set KIT/SET CONTAINS: 1 vial (50 µg) LAG-3:Fc (human) (recom-

binant), 1 vial (100 µg) MAb to LAG-3 (human) (17B4) (Biotin) [Detection Antibody], 1 vial (100 µg) MAb to LAG-3 (human)

(11E3) [Capture Antibody]. APPLICATION: ELISA.LIT: A soluble lymphocyte activation gene-3 (sLAG-3) protein as aprognostic factor in human breast cancer expressing estrogen orprogesterone receptors: Triebel, et al.; Cancer Lett. 2005 (in press)

FIGURE: Detection of endogenous human LAG-3 by FACS analysisusing the MAb to LAG-3 (17B4) (human) (ATTO 488) (Prod. No.ALX-804-806TD). METHOD: Human PBMC were stimulated (right)or not (left) with 1 µg/ml of superantigen SEB. After two days,PBMC were stained with 10 µg/ml (1µg/0.5x106 cells) of MAb toLAG-3 (17B4) (ATTO 488) and analyzed by Flow Cytometry.

L A B E L L E D A N T I B O D I E S

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DRAQ5™ A cell-permeant far red-fluorescing DNA probe

New Antibodies – Latest Additions

PAb to Cornulin (human) (SZ1229)ALX-210-922-C050 50 µgFrom rabbit. IMMUNOGEN: Synthetic peptide correspondingto aa 259-274 (E259ATNDQNRGTETHGQG9 274) of human cor-nulin. SPECIFICIT Y: Recognizes human cornulin. Detects a band of ~70kDa by Western blot. APPLICATION: IHC (FS),WB.LIT: Cornulin, a new member of the „fused gene“ family, is ex-pressed during epidermal differentiation: R. Contzler, et al.; J. Invest. Dermatol. 124, 990 (2005)

PAb to CTRP2 (mouse) (AT102)ALX-210-923-C050 50 µgFrom rabbit. IMMUNOGEN: Recombinant mouse CTRP2 (aa 26-260). SPECIFICIT Y: Recognizes mouse CTRP2. APPLICATION: IP, WB.

PAb to CTRP7 (mouse) (AT103)ALX-210-924-C050 50 µgFrom rabbit. IMMUNOGEN: Recombinant mouse CTRP7 (aa 18-290). SPECIFICIT Y: Recognizes mouse CTRP7. APPLICATION: IP, WB.

PAb to Nerve Growth FactorReceptor (human) (AT101)ALX-210-921-C050 50 µgFrom rabbit. IMMUNOGEN: Recombinant human NGFR:Fc(Prod. No. ALX-522-044). The extracellular domain of hu-man nerve growth factor receptor (NGFR) (aa 1-264) is fusedto the Fc portion of human IgG1. SPECIFICIT Y: Recognizes hu-man NGFR. APPLICATION: WB.

MAb to RANKL (human) (Ranky-1)ALX-804-830-C100 100 µgCLONE: Ranky-1. ISOT YPE: Rat IgG1. IMMUNOGEN: Recom-binant human soluble RANKL (aa 151-316) (Prod. No. ALX-522-012). The extracellular domain of human RANKL (aa 151-316) is fused at the N-terminus to a linker peptide (6aa) and a FLAG®-tag. SPECIFICIT Y: Recognizes human RANKL.APPLICATION: ELISA, WB.

PAb to Repetin (AF646)ALX-210-925-C050 50 µgFrom rabbit. IMMUNOGEN: Synthetic peptide correspondingto aa 570-584 (H570YGQTDRQGQS SHYI584) of human repe-tin. SPECIFICIT Y: Recognizes human and mouse repetin. De-tects a band of ~100kDa by Western blot. APPLICATION: IHC(FS), WB.LIT: Isolation and characterization of human repetin, a member of the fused gene family of the epidermal differentiation com-plex: M. Huber, et al.; J. Invest. Dermatol. 124, 998 (2005)

MAb to SARM (human) (Sarmy-1) ALX-804-831-C100 100 µgCLONE: Sarmy-1. ISOT YPE: Mouse IgG2b. IMMUNOGEN: Re-combinant human SARM (aa 93-292). SPECIFICIT Y: Recog-nizes human SARM. APPLICATION: IP.

Thy-1 (human):Fc (human) (rec.) ALX-522-091-C050 50 µgProduced in HEK 293 cells. The extracellular domain of hu-man Thy-1 (aa 20-130) is fused to the Fc portion of hu-man IgG1.

BOS-889-001-R050 50 µlBOS-889-001-R200 200 µlDRAQ5™ is a highly cell permeable DNA-interactive agent, with fl uorescence signature extending into the infrared region of the spectrum. The unique characteristics of DRAQ5™ offer excit-ing new advantages for investigations using both fi xed speci-mens and viable cells.Manufactured by Biostatus Ltd. DRAQ5™ is a registered trademark of Biostatus Ltd.

FIGURE: Triple-label false color images using: a mitochondrion-specific dye (red), a GFP-labelled fusion protein (green) and DRAQ5™ usedas a vital DNA dye (arbitrarily assigned the color blue). Cells shown (from left to right): Interphase breast tumor cells, anaphase breast tumorcells and metaphase breast tumor cells.

... procedure is relatively short and simple; the qual-p y p qity of staining is reliable; and technically, the meas-y g yurement can be easily handled. These advantageousy gfeatures make DRAQ5™ excellent for additional pro-pspective multicentric testing as previously recommend-p g p yed for clinical diagnosis and immunophenotyping ofghematopoetic malignancies.” Optimization of three- and four-color multiparameter DNA analysis in lymphoma speci-pmens: M. Plander, et al.; Cytometry A y y 54, 66 (2003) “Because of its simplicity of use, excitability with 488 nm lasers, p y yand the ability to stain viable cells, DRAQ5 should provey pmost useful in the kinetic evaluation of normal and neoplastic hematolymphoid cell subsets identified by p y plight scatter and antigenic expression.” DRAQ5-basedDNA content analysis of hematolymphoid cell subpopulations discriminated by surface antigens and light scatter properties: pC.M. Yuan, et al.; Cytometry B Clin. Cytom.y y y 58, 47 (2004) ”... we demonstrate how we incorporate the smart design of a p gtwo-photon ‚dark‘ DNA binding probe, such as DRAQ5, asp g pa well-defined quenching probe to uncover sites of druginteraction ”.” Advanced microscopy solutions for monitoring the kinetics and dynamics of drug-DNA targeting in living cells:R.J. Errington, et al.; Adv. Drug Deliv. Rev. 57, 153 (2005)

Key Literature & TestimonialsA novel cell permeant and far red-fluorescing DNA probe, DRAQ5, for blood cell discrimination by flow cytometry: P.J. Smith, et al.; J. Immunol. Meth. 229, 131 (1999)

TABLE: Optimal excitation and emission wavelengths for DRAQ5™ and other fluorophores.

FIGURE: Spectralcharacteristics of DRAQ5™ Emission

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