Labeling and Detection

The best and brightestAlexa Fluor® 488 dye

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Labeling and Detection

A superior alternative to FITCBrighter conjugate fluorescence →

Unequalled photostability →

Perfect spectral match for FITC filters →

Now you have a choice when it comes to green-fluorescent dye conjugates. Molecular

Probes™ Alexa Fluor® 488 dye—with nearly identical spectral properties and quantum

yield as fluorescein (FITC)—produces brighter, more photostable conjugates that are ideal

for imaging and other applications requiring increased sensitivity and environmentally

insensitive fluorescence detection.

High performance green-fluorescent dye conjugatesFITC is the most commonly used fluorophore in many biological research areas. The fluo-

rophore has much to recommend it, such as visible light excitation and emission, a high

quantum yield, and pH sensitivity in the physiological range. With significant advances in

detection technology, however, the limitations of FITC have become apparent:

Collisional quenching of FITC fluorescence when multiple dyes are attached to a pro- →

tein or small molecule

Fast rates of FITC photobleaching upon exposure to excitation light →

Quenching of the FITC fluorescence under slightly acidic conditions →

These limitations prevent the researcher from getting the brightest conjugates, the most

sensitive fluorescence signal, and flexibility in buffering conditions.

Figure 1—Comparison of the relative fluorescence of Alexa Fluor® 488 dye and FITC. Goat anti–mouse IgG conjugate fluorescence was determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of a reference dye and multiplying by the dye:protein labeling ratio.

Con

juga

te fl

uore

scen

ce

Fluorophores/protein (mol:mol)0 2 6 8 104

Alexa Fluor® 488

FITC

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Advantages of the Alexa Fluor® 488 dye“All of the Alexa Fluor® dyes are superior to anything out there. [They are the] best reagents

since sliced bread. FITC has been banned from this lab.” —Joe Goodhouse, Department of

Molecular Biology, Princeton University

Brighter conjugate fluorescence

Fluorescein conjugates rapidly quench as more fluorophores are added. The

Alexa Fluor® 488 dye allows more fluorophores to be attached to the conjugate before

self-quenching becomes apparent, leading to significantly brighter conjugates (Figure 1).

This increased brightness means that you can use less conjugate in your experiments,

reducing background fluorescence and stretching your research dollar.

Unequalled photostability

The superior photostability of the Alexa Fluor® 488 dye allows more time for image obser-

vation and capture, thereby permitting greater sensitivity and simplifying the detection of

low-abundance targets (Figure 2).

Perfect spectral match for FITC filters

The absorption and emission profiles of Alexa Fluor® 488 dye are nearly identical to those

of FITC (Figure 3)—no need to change equipment, settings, or filters.

Figure 2—Photobleaching profiles of cells stained with Alexa Fluor® 488 or fluorescein. Alexa Fluor® 488 dye and fluorescein conjugates of goat anti–mouse IgG antibody F(ab’)2 fragment were used to detect HEp-2 cells probed with human anti-nuclear antibodies. Samples were continuously illuminated and images were collected every 5 seconds with a cooled CCD camera. Normalized intensity data demonstrate the difference in pho-tobleaching rates.

Figure 3—Absorption and fluorescence emission spectra of fluorescein and Alexa Fluor® 488 dye. The fluorescence intensity of the Alexa Fluor® 488 goat anti–mouse IgG conjugate (—) was signifi-cantly higher than that of the fluorescein goat anti–mouse IgG conjugate (---). The data are nor-malized to show the spectral similarity.

®

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Labeling and Detection

A broad spectrum of Alexa Fluor® 488 dye productsSecondary antibodies and streptavidin

Our species-specific anti-IgG antibod-

ies are affinity purified and adsorbed

against the sera of a number of species

to minimize cross-reactivity. Anti-IgM

conjugates are prepared from well-char-

acterized antibodies that have been puri-

fied by IgM affinity chromatography and

react specifically with IgM heavy chains

(Table 1). We also offer an Alexa Fluor®

488 dye–labeled streptavidin conjugate

for detection of endogenous biotin or

biotinylated targets.

Tyramide signal amplification kits

Tyramide signal amplification (TSA) tech-

nology is an enzyme-mediated detection

method that utilizes the catalytic activity

of horseradish peroxidase (HRP) to gener-

ate high-density labeling of a target pro-

tein or nucleic acid sequence in situ. The

TSA method has been reported to increase

sensitivity up to 100-fold compared with

conventional avidin–biotinylated enzyme

Table 1—Secondary antibody and streptavidin conjugates.

Host Target Cat. no.

Goat Mouse IgG A11001, A11017,* A11029†

Rabbit Mouse IgG A11059, A21204†

Chicken Mouse IgG A21200

Donkey Mouse IgG A21202

Goat Mouse IgG1 A21121

Goat Mouse IgG2a A21131

Goat Mouse IgG2b A21141

Goat Mouse IgG3 A21151

Goat Mouse IgM A21042

Goat Rabbit IgG A11008, A11070,* A11034†

Chicken Rabbit IgG A21441

Donkey Rabbit IgG A21206

Goat Chicken IgY A21441

Goat Guinea pig IgG A11073

Goat Hamster IgG A21110

Goat Human IgG A11013

Goat Human IgM A21215

Goat Rat IgG A11006

Chicken Rat IgG A21470

Donkey Rat IgG A21208

Rabbit Rat IgG A21210

Goat Rat IgM A21212

Donkey Sheep IgG A11015

Rabbit Goat IgG A11078, A21222*

Chicken Goat IgG A21467

Donkey Goat IgG A11055

Streptavidin‡ Biotin A11223, A32354

* F(ab’)2 fragment. † Adsorbed against additional species. ‡ Available lyophilized or in solution.

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complex (ABC) procedures. The signal amplification conferred by the turnover of multiple

Alexa Fluor® 488 labeled tyramide substrates per peroxidase label translates into practical

benefits, namely ultrasensitive detection of low-abundance targets in fluorescence in situ

hybridization, immunohistochemistry, and other applications (Table 2).

Zenon® labeling technologyZenon® labeling kits (Table 3) provide a versatile and easy-to-use method for labeling

antibodies, even with very small (submicrogram) amounts of starting material. Zenon®

antibody labeling technology forms a labeling complex using a fluorophore-labeled Fab

fragment that is selective for the Fc portion of a primary antibody. Simple mixing of the

labeled Fab fragment with an intact primary antibody rapidly and quantitatively forms the

labeling complex. This labeling complex is then used for staining in the same manner as

a covalently labeled primary antibody. Think of Zenon® labeling technology as “staining

with your secondary first”.

Table 3—Zenon® labeling kits.

Target antibody species/isotype Cat. no.

Mouse IgG1 Z25002

Mouse IgG2a Z24102

Mouse IgG2b Z25202

Rabbit IgG Z25302

Goat IgG Z25602

Human IgG Z25402

Table 2—Tyramide signal amplification kits.

Peroxidase conjugate Cat. no.

Goat anti–mouse IgG T20912

Goat anti–rabbit IgG T20922

Streptavidin T20932

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Labeling and Detection

Table 5—Protein labeling kits.

Labeling kitQuantity labeled

per reactionNo. of

reactionsCat. no.

Protein Labeling Kit 1 mg 3 A10235

Monoclonal Antibody Labeling Kit 100 µg 5 A20181

Microscale Protein Labeling Kit 20–100 µg 3 A30006

Conjugates for a variety of applicationsInvitrogen offers a wide selection of Alexa Fluor® 488 dye–labeled protein and small mole-

cule conjugates for a variety of applications (Table 4). Each of these conjugates outperforms

the comparable fluorescein conjugate to give you the best results for your experiments.

Do-it-yourself labelingOur Alexa Fluor® 488 protein labeling kits combine years of labeling experience with the

superior performance of our Alexa Fluor® 488 dye. All materials are provided, including

those for purification, and the entire procedure takes about two hours, with little hands-

on time. Several types of labeling kits are available (Table 5), including general protein

labeling kits, microscale protein labeling kits (for small amounts), and monoclonal anti-

body labeling kits (optimized for labeling antibodies).

Table 4—Protein and small molecule conjugates.

Conjugate or small molecule Application/function Cat. no.

Phalloidin Cytoskeletal probe A12379

Actin (rabbit muscle) Cytoskeletal dynamics A12373

Transferrin Endocytosis T13342

Epidermal growth factor (EGF) Endocytosis E13345

Annexin V Apoptosis A13201

Cholera toxin, subunit B (CT-B) Lipid rafts C34775

Wheat germ agglutinin (WGA) Carbohydrate probe W11261

Concanavalin A (Con A) Carbohydrate probe C11252

Isolectin IB4 Carbohydrate probe I21411

Hydrazide Gap junctions, tracing A10436

Dextran (3,000 MW) Tracing D34682

Dextran (10,000 MW) Tracing D22910

Anti-BrdU mouse monoclonal IgG TUNEL, cell proliferation A21303

Bovine serum albumin Cell tracing, endocytosis A13100

Ovalbumin Cell tracing, endocytosis O34781

Low-density lipoprotein from human plasma, acetylated

Endocytosis L23380

α-Bungarotoxin Neuromuscular junctions B13422

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Intermediate filaments of astrocytes and epen-dymal cells in a 14 µm mouse brain cryosection. Filaments were labeled using mouse monoclonal anti–glial fibrillary acidic protein antibody (anti-GFAP) and visualized with green-fluorescent Alexa Fluor® 488 goat anti–mouse IgG antibody. Nuclei were stained with blue-fluorescent DAPI. The im-age was deconvolved using Huygens software (Scientific Volume Imaging); 3D reconstruction was performed using Imaris software (Bitplane AG).

The peripheral nervous system of a wild-type (Canton-S) Drosophila melanogaster embryo. The embryo was labeled with the monoclonal 22c10 antibody (which detects a microtubule-associated protein) and subsequently visualized using green-fluorescent Alexa Fluor® 488 rabbit anti–mouse IgG antibody. The actively dividing cells of the devel-oping denticle bands were labeled with a rabbit anti–histone-H3 antibody and visualized using red-fluorescent Alexa Fluor® 594 goat anti–rabbit IgG antibody. Finally, the nuclei, which are concentrated in the central nervous system, were counterstained with blue-fluorescent DAPI. Image contributed by Neville Cobbe, University of Edinburgh.

Hippocampal region of a mouse brain cryosection. Capillaries were visualized with the green-fluores-cent Alexa Fluor® 488 conjugate of lectin HPA from Helix pomatia, which specifically binds to type-A erythrocytes and a-N-acetylgalactosaminyl resi-dues. The nuclei were counterstained with nuclear yellow. The multiple-exposure image was acquired using a DAPI longpass filter set and a filter set ap-propriate for fluorescein.

The cytoskeleton of a fixed and permeabilized bo-vine pulmonary artery endothelial cell. Tubulin was detected using mouse monoclonal anti–α-tubulin antibody and visualized with Alexa Fluor® 647 goat anti–mouse IgG antibody (pseudocolored magen-ta). Endogenous biotin in the mitochondria was labeled with green-fluorescent Alexa Fluor® 488 streptavidin; DNA was stained with blue-fluores-cent DAPI.

Mouse intestine cryosection. Basement membranes were labeled with chicken IgY anti-fibronectin anti-body and visualized using green-fluorescent Alexa Fluor® 488 goat anti–chicken IgG. Goblet cells and crypt cells were labeled with red-fluorescent Alexa Fluor® 594 wheat germ agglutinin. The microvil-lar brush border and smooth muscle layers were visualized with Alexa Fluor® 680 phalloidin (pseu-docolored purple); nuclei were stained with blue-fluorescent DAPI.

Fixed, permeabilized muntjac skin fibroblast. Mi-tochondria were labeled with anti–OxPhos Com-plex V inhibitor protein mouse IgG1 and visualized using orange-fluorescent Alexa Fluor® 555 goat anti–mouse IgG. F-actin was labeled with green-fluorescent Alexa Fluor® 488 phalloidin; the nucleus was stained with TO-PRO®-3 stain (pseudocolored magenta).

For more information about the Alexa Fluor® 488 dye and related products, please visit www.invitrogen.com/probes.

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©2006 Invitrogen Corporation. All rights reserved. These products may be covered by one or more Limited Use Label Licenses (see Invitrogen catalog or www.invitrogen.com). By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated. B-068179-r1 1106