the best and brightest - unicaen · the best and brightest alexa fluor ... alexa 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