Labeling and Detection The best and brightest Alexa Fluor 488 dye
Labeling and Detection A superior alternative to FITC Brighter 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 conjugates FITC is the most commonly used fluorophore in many biological research areas. The fluorophore 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- Conjugate fluorescence Alexa Fluor 488 FITC tein or small molecule Fast rates of FITC photobleaching upon exposure to excitation light 0 2 4 6 8 10 Fluorophores/protein (mol:mol) 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. 2
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 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 antinuclear antibodies. Samples were continuously illuminated and images were collected every 5 seconds with a cooled CCD camera. Normalized intensity data demonstrate the difference in photobleaching rates. 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 observation 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 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 significantly higher than that of the fluorescein goat anti mouse IgG conjugate (---). The data are normalized to show the spectral similarity. www.invitrogen.com 3
Labeling and Detection A broad spectrum of Alexa Fluor 488 dye products Secondary antibodies and streptavidin Our species-specific anti-igg antibodies are affinity purified and adsorbed against the sera of a number of species to minimize cross-reactivity. Anti-IgM conjugates are prepared from well-characterized antibodies that have been purified 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) technology is an enzyme-mediated detection method that utilizes the catalytic activity of horseradish peroxidase (HRP) to generate high-density labeling of a target protein 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. 4
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 technology Zenon 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 2 Tyramide signal amplification kits. Peroxidase conjugate Goat anti mouse IgG Goat anti rabbit IgG Streptavidin Cat. no. T20912 T20922 T20932 Table 3 Zenon labeling kits. Target antibody species/isotype Mouse IgG1 Mouse IgG2a Mouse IgG2b Rabbit IgG Goat IgG Human IgG Cat. no. Z25002 Z24102 Z25202 Z25302 Z25602 Z25402 www.invitrogen.com 5
Labeling and Detection Conjugates for a variety of applications Invitrogen offers a wide selection of Alexa Fluor 488 dye labeled protein and small molecule 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 labeling Our 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 handson time. Several types of labeling kits are available (Table 5), including general protein labeling kits, microscale protein labeling kits (for small amounts), and monoclonal antibody 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 IB 4 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 Table 5 Protein labeling kits. Labeling kit Quantity labeled per reaction No. of reactions Cat. 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 6
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 greenfluorescent Alexa Fluor 488 rabbit anti mouse IgG antibody. The actively dividing cells of the developing denticle bands were labeled with a rabbit anti histone-h3 antibody and visualized using redfluorescent 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-fluorescent Alexa Fluor 488 conjugate of lectin HPA from Helix pomatia, which specifically binds to type-a erythrocytes and a-n-acetylgalactosaminyl residues. The nuclei were counterstained with nuclear yellow. The multiple-exposure image was acquired using a DAPI longpass filter set and a filter set appropriate for fluorescein. Mouse intestine cryosection. Basement membranes were labeled with chicken IgY anti-fibronectin antibody 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 microvillar brush border and smooth muscle layers were visualized with Alexa Fluor 680 phalloidin (pseudocolored purple); nuclei were stained with bluefluorescent DAPI. Intermediate filaments of astrocytes and ependymal cells in a 14 µm mouse brain cryosection. Filaments were labeled using mouse monoclonal anti glial fibrillary acidic protein antibody (antigfap) and visualized with green-fluorescent Alexa Fluor 488 goat anti mouse IgG antibody. Nuclei were stained with blue-fluorescent DAPI. The image was deconvolved using Huygens software (Scientific Volume Imaging); 3D reconstruction was performed using Imaris software (Bitplane AG). The cytoskeleton of a fixed and permeabilized bovine 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 magenta). Endogenous biotin in the mitochondria was labeled with green-fluorescent Alexa Fluor 488 streptavidin; DNA was stained with blue-fluorescent DAPI. Fixed, permeabilized muntjac skin fibroblast. Mitochondria were labeled with anti OxPhos Complex V inhibitor protein mouse IgG1 and visualized using orange-fluorescent Alexa Fluor 555 goat anti mouse IgG. F-actin was labeled with greenfluorescent 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. 7 www.invitrogen.com
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