Superior Fluorescent Labeling Dyes Spanning the Full Visible Spectrum...1. Trademarks: HiLyte Fluor (AnaSpec, Inc.)

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2 Table of Contents Fluor TM Labeling Dyes Superior Fluorescent Labeling Dyes Spanning the Full Visible Spectrum....1 Fluor TM 405 Dye, an Excellent Alternative to Alexa Fluor 405 & DyLight Fluor TM 488 Dye, an Excellent Alternative to FITC...3 Fluor TM 532 Dye, an Excellent Alternative to Cy3 TM Dye...4 Fluor TM 555 Dye, an Excellent Alternative to Cy3 TM Dye...5 Fluor TM 647 Dye, an Excellent Alternative to Cy5 TM Dye...6 Fluor TM 750 Dye, an Excellent Alternative to Cy7 TM Dye...8 AnaTag TM Fluor TM Protein Labeling Kits...9 Fluor TM Labeled Primary and Secondary Antibodies.... Back Cover Fluor TM Labeled Streptavidins...Back Cover Trademarks: Fluor (AnaSpec, Inc.) Alexa Fluor, Texas Red (Invitrogen) Cy (GE Healthcare) DyLight Fluor (Pierce)

3 Superior Fluorescent Labeling Dyes Spanning the Full Visible Spectrum Fluor dyes, AnaSpec's patented dyes are a series of excellent fluorescent labeling dyes that span the full visible spectrum. For most of the Fluor dyes, their hydrophilic property makes conjugations to protein easier, minimizing the use of organic solvents. The resulting conjugates are relatively resistant to precipitation during storage. The Fluor dyes also have better labeling performance than the classic fluorescent labeling dyes such as FITC and rhodamines. Other features of the Fluor dyes include: Fluor dyes are available in a variety of reactive forms. Fluor conjugates exhibit more intense fluorescence than other spectrally similar conjugates of classic fluorescent dyes such as FITC, TAMRA and ROX under similar excitations. Fluor dyes are more photostable than the classic fluorescent dyes such as FITC, Cy3 and Cy5. Fluor dyes have absorption spectra that match the principal output wavelengths of common excitation sources such as 488 nm, 555 nm, 633 nm and 647 nm. Fluor dyes and their conjugates are available in several distinct fluorescent colors. Fluor dyes are highly fluorescent over a broad ph range with little ph sensitivity. Fluor dyes are the products of our R&D efforts. Additionally, AnaSpec is rapidly expanding our product lines to meet research needs. We have been developing dyes to solve various limitations with the existing fluorescent labeling reagents while offering classic fluorescent labeling reagents which have high purity and competitive pricing. We also provide custom synthesis and contract research services to meet your special needs. Our strong expertise in both the chemistry of fluorescent dyes and peptides equip us to provide quality services with competitive prices Fluor Fluor Fluor Fluor Fluor Fluor Fluor Fluor Fluor Fluor Fluor Fluor 750 Normalized Emission Figure 1. Absorption and emission spectra of AnaSpec's patented Fluor dyes. 1

4 Fluor 405 Dye, an Excellent Alternative for Alexa Fluor 405 and DyLight 405 Fluor 405, available in a variety of reactive forms, such as amine-reactive, thiol-reactive and amine-containing derivatives, allows for easy conjugation to biopolymers. Fluor 405 has spectral characteristics similar to those of Alexa Fluor 405 and DyLight 405 dyes with excitation and emission wavelength at 404/428 nm in PBS buffer (ph 7.4). Conjugates of Fluor 405 are well excited by the 407 nm spectral line of the Krypton laser and the 408 nm line of the violet laser diode. Fluor 405, whether conjugated to primary tag, secondary antibodies or streptavidin, are prepared in an optimized fluorophore/protein labeling ratio to ensure high fluorescent signal and uncompromised biological function. Extinction coefficient is 35,000 M -1 cm -1, with a quantum yield of Product Size Cat# Fluor 405 hydrazide 1 mg Fluor 405 succinimidyl ester (SE) 1 mg Normalized Fluorescence Figure 3. Alpha-tubulin in 3T3 cells probed with mouse anti-a tubulin antibody, visualized with Fluor 405 conjugated goat anti-mouse IgG 2

5 Fluor 488 Dye, an Excellent Alternative for FITC Although FITC is still the most popular fluorescent labeling dye for preparing green fluorescent bioconjugates, there are limitations, such as severe photobleaching for microscope imaging and ph-sensitive fluorescence. Protein conjugates prepared with Fluor 488 dyes (Ex/Em=497/525 nm) are far more superior compared to conjugates of fluorescein derivatives such as FITC (Figure 2). Fluor 488 conjugates are significantly brighter than fluorescein conjugates and are much more photostable. Extinction coefficient is 70,000 M -1 cm -1, with a quantum yield of Additionally, the fluorescence of Fluor 488 is not affected by ph (4-10). This ph insensitivity property is a major improvement over fluorescein, which emits maximum fluorescence only at ph above 9. Product Size Cat# Fluor 488 acid 10 mg Fluor 488 amine, TFA salt 1 mg Fluor 488 C2 maleimide 1 mg Fluor 488 hydrazide 1 mg Fluor 488 succinimidyl ester (SE) 5 mg A Normalized Fluorescence B Fluor C Absorbance FITC % Original Fluorescence Fluor FAM 96 FITC Irradiation Time (min) Time (sec) Figure 4. Absorption and emission spectra of Fluor 488 (panel A). Panels B and C show the photostability of Fluor 488 compared to FITC and FAM. Figure 5. Tubulin in human glioblastoma cells was probed with mouse anti-tubulin, visualized with Fluor 488-conjugated rabbit anti-mouse IgG. Nuclei were stained with Hoechst (Cat# 83218). 3

6 Fluor 555 Dye, an Excellent Alternative for Cy3 Dye Although Cy3 is the preferred dye for preparing orange fluorescent bioconjugates, Fluor 555 conjugates are more photostable and brighter. Compared to the spectra of Cy3 dyes, the spectra of Fluor 555 conjugates are slightly red-shifted, resulting in an optimal match of filters designed for Cy3 dyes. Extinction coefficient is 150,000 M -1 cm -1, with a quantum yield of The photostability of Fluor 555 provides researchers with additional time for image capture. Product Size Cat# Fluor 555 acid 5 mg Fluor 555 amine 1 mg Fluor 555 C2 maleimide 1 mg Fluor 555 succinimidyl ester (SE) 1 mg A Normalized Fluorescence Emission FLuor 555 Cy3 B Label Ratio (Dye:Protein) C Figure 6. Absorption and emission spectra of Fluor 555 (panel A). Comparison data of Fluor 555 and Cy3 emission at different dye to protein ratio (panel B). 3T3 cells incubated with anti-tubulin antibody and goat-anti-rabbit antibodies, labeled either with Fluor 555 (panel C), or Cy3 (panel D). Nuclei were stained with Hoechst (Cat# 83218). D Figure 7. Bovine pulmonary artery endothelial cells actins were stained with biotin-conjugated phalloidin, visualized with Fluor 555 conjugated streptavidin (Cat# 60666). Mitochondria were stained with mouse anti-oxphos complex V, visualized with Fluor 488- conjugated goat anti-mouse, nuclei labeled with Hoechst (Cat# 83218). 4

7 Fluor 647 Dye, an Excellent Alternative for Cy5 Dye Cy5 is the preferred dye for preparing near-infrared fluorescent bioconjugates. Compared to the spectra of Cy5 dyes, the spectra of Fluor 647 conjugates are only slightly red-shifted. This slight change in absorption spectrum makes Fluor 647 dyes an optimal match of filters designed for Cy5 dyes. In a side-by-side comparison of antibody conjugates of Fluor 647 dyes and Cy5 conjugates (supplied by other companies), the total fluorescence of Fluor 647 labeled secondary antibodies is significantly higher than that of Cy5 conjugates. Extinction coefficient is 250,000 M -1 cm -1, with a quantum yield of Unlike Cy5 dyes, Fluor 647 dyes have very little change in absorption or fluorescence spectra when conjugated to most proteins, oligonucleotides and nucleic acids, thus yielding greater total fluorescence at the same degree of substitution. Product Size Cat# Fluor 647 acid 5 mg Fluor 647 amine 1 mg Fluor 647 C2 maleimide 1 mg Fluor 647 succinimidyl ester (SE) 1 mg A Normalized Fluorescence RFU Fluor Alexa Fluor 647 DyLight Cy Rabbit IgG (ng/well) B A/A 0 C Fluor TM 647 Alexa Fluor TM Irradiation Time (min) DC DE Figure 9. Absorption and emission spectra of Fluor 647 (panel A). Performance of dye-goat anti-rabbit IgG conjugates (panel B). Fluor 647 and Alexa Fluor 647 photostability over time (panel C). 3T3 cells incubated with anti-tubulin antibody and goat-anti-rabbit antibodies, labeled either with Fluor 647 (panel D), or Cy5 (panel E), nuclei were stained with Hoechst (Cat# 83218). Figure 10. a-tubulins of bovine pulmonary artery endothelial cells were probed with biotin-conjugated mouse anti-a-tubulin, visualized with Fluor 647 conjugated streptavidin (Cat# 60667). Mitochondria were detected with mouse anti-oxphos V complex, visualized with Fluor 488-conjugated goat anti-mouse, nuclei stained with DAPI (Cat# 83210). 6

8 Fluor 750 Dye, an Excellent Alternative for Cy7 Dye Spectrally similar to Cy7 dye, Fluor 750 dye is the longest-wavelength Fluor dye currently available. Its fluorescence emission maximum at 778 nm is well separated from commonly used far-red fluorophores, including Fluor 647, or allophycocyanin (APC), facilitating multicolor analysis. Extinction coefficient is 275,000 M -1 cm -1, with a quantum yield of With a peak excitation at ~753 nm, conjugates of Fluor 750 dyes can be excited by a xenon-arc lamp or dye-pumped lasers operating in the nm range. Product Size Cat# Fluor 750 amine 1 mg Fluor 750 C2 maleimide 1 mg Fluor 750 hydrazide 1 mg Fluor 750 succinimidyl ester (SE) 1 mg Normalized Fluorescence Figure 13. Absorption and emission spectra of Fluor 750 (top). Bottom image shows an image of CHO-K1 cells labeled with rabbit anti-tubulin primary antibody and a secondary antibody goat anti-rabbit IgG conjugated to Fluor 750 (red); Paxillin EGFP (green) and a nuclear stain, DAPI (blue). Image courtesy of Kimberly Young and Claire M. Brown, McGill University Imaging Facility, Montreal, Canada. 8

9 AnaTag Protein Labeling Kits The AnaTag Fluor Protein Labeling Kits provide a convenient way to label proteins using the succinimidyl ester (SE) reactive form of the Fluor dyes. The succinimidyl ester shows good reactivity and selectivity with aliphatic amines of the protein and forms a carboxamide bond, which is identical to, and is as stable as the natural peptide bond. Fluor -protein conjugates may be used in immunofluorescent staining, fluorescent in situ hybridization, flow cytometry and other biological applications. Each kit has all the essential components for performing the conjugation reaction and for purifying the Fluor -protein conjugate. Figure 14. The succinimidyl ester (SE) group of the fluorophore reacts with the amino group of lysines on the protein to form a stable carboxamide bond. Table 1. AnaTag Fluor Protein Labeling kits are available in large scale (3x5 mg) or microscale (3x200 ug) sizes. Product Abs / Em (nm / nm) 3 x 5 mg 3 x 200 ug AnaTag Fluor 488 Protein Labeling Kit 499 / AnaTag Fluor 555 Protein Labeling Kit 553 / AnaTag Fluor 647 Protein Labeling Kit 652 / AnaTag Fluor 750 Protein Labeling Kit 754 / Figure 15. Labeling of an amino group (for instance, side chain of lysine) on a biopolymer with a succinimidyl ester of a dye in 4 easy steps using the AnaTag Protein Labeling Kits. 9

10 Fluor Labeled Primary and Secondary Antibodies AnaSpec's Fluor dye-igg conjugates are prepared using optimal fluorophore to protein labeling ratio to ensure high fluorescent signal and uncompromised IgG function. These Fluor labeled primary (Table 2) and secondary antibodies (Table 3) can be used in applications such as fluorescence microscopy and flow cytometry. AnaSpec also provides labeling service of our catalog or custom antibodies. Labels include the Fluor dyes or the classic dyes, such as FITC and TAMRA. Table 2. Examples of some Fluor labeled primary antibodies. Product Fluor 488 Fluor 555 Fluor 647 Anti-a-Actin (smooth muscle), polyclonal H H H647 Anti-GST Tag, monoclonal H H H647 Anti-His Tag, polyclonal H H H647 Anti-His Tag, monoclonal H H H647 Fluor Labeled Secondary Antibodies Sampler Kits Table 3. These sampler kits contain 6 different Fluor labeled secondary antibodies - Fluor 488, 555, and 647 labeled goat anti-mouse IgGs and goat anti-rabbit IgGs. Size Goat anti-mouse IgG Goat anti-rabbit IgG Goat anti-mouse IgG Goat anti-rabbit IgG (highly crossadsorbed) Rabbit anti-mouse IgG Rabbit anti-goat IgG 6 x 50 ug Fluor Streptavidin Conjugates Table 4. A listing of Fluor -streptavidin conjugates. Product Fluor 488 Fluor 555 Fluor 647 Fluor 750 Streptavidin H750 Fluor Labeled Streptavidin Sampler Kits Table 5. These sampler kits contain streptavidin conjugates of Fluor -488, 555 and 647. Size Fluor Labeled Streptavidin Sampler Kits 3 x 20 ug

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