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SaraFluor™ Series

SaraFluor™ 650

[Fluorophores for labeling]

650-750 nm：Far red

SaraFluor series is a product line of bright fluorophores, which are suitable for labeling proteins (e.g., antibodies) and other macromolecules. Among them, SaraFluor 650, 700, and 720, derivatives of silicone rhodamine, are uniquely bright and photostable fluorophores in far-red to near-infrared wavelength range.
N-hydroxysuccinimide (NHS) esters quickly form a covalent bond with primary amine just by mixing. NHS esters are widely used for labeling antibodies and macromolecules. On the other hand, maleimides which forms a covalent bond with thiols (R-SH) are used to specifically label cysteine residues. The fluorophores with carboxyl groups (-COOH) are used for labeling using crosslinkers, used as a material for chemical synthesis.

SaraFluor is named after an Ainu language word “sara” which means “become visible” or “spacious and bright wetland”.

Products

Code No. Product Name Size Merck CAT No. Merck ( Millipore / Sigma Aldrich )
Product Name
ST1008-10 SaraFluor™ 650-NHS 5 nmol x 5
ST1008-11 SaraFluor™ 650-NHS 1 mg
ST1008-15 SaraFluor™ 650-NHS 1 mg x 5
ST1008-21 SaraFluor™ 650-maleimide 1 mg
ST1008-25 SaraFluor™ 650-maleimide 1 mg x 5
ST1008-31 SaraFluor™ 650-COOH 1 mg
ST1008-35 SaraFluor™ 650-COOH 1 mg x 5

Downloads

  • SaraFluor-NHS Protocol

  • Flyer

  • ST1008-10,11,15 SDS

  • ST1008-21,25 SDS

  • ST1008-31,35 SDS

  • ST1008-41,45 SDS

  • Product Information

    SaraFluor™ 650
    Product Information

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    Properties of SaraFluor series

    Product name Absmax (nm) FLmax (nm) ε (M-1 cm-1) Φ Similar fluorophores in the same wavelength range
    SaraFluor 488 494 514 0.82 × 105 0.91 FITC, Alexa Fluor 488, ATTO 488
    SaraFluor 600 597 607 1.3 × 105 0.48 Cy3.5, Texas Red, Alexa Fluor 594
    SaraFluor 650 646 660 1.1 × 105 0.31 Cy5, Alexa Fluor 647, ATTO 647N
    SaraFluor 700 691 712 1.0 × 105 0.12 Cy5.5, Alexa Fluor 700, ATTO 700
    SaraFluor 720 721 740 1.6 × 105 0.05 Cy7, DY-730, ATTO 725

    Spectra

    Spectra of SaraFluor series (dot lines : absorption spectra, solid lines : emission spectra)

     

    Photostability

    A comparison of photobleaching property of SaraFluor 650

    Fixed cells were stained with secondary antibodies labeled with SaraFluor 650 or another corresponding fluorophore, and were observed by fluorescence microscopy. Photobleaching was observed under the same imaging conditions including light intensity, fluorescent filters, exposure, and camera gain.

     

  • Images using SaraFluor series

    SaraFluor™ 650
    Images using SaraFluor series

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    Images using SaraFluor series

    An image using SaraFluor 488-NHS

    OVCAR5 cells fixed and permeabilized with 1% Triton-X100 were reacted with rabbit anti-α/β tubulin antibody (#2148, Cell Signaling Technology 1/50) and then, reacted with goat anti-rabbit antibody labeled using SaraFluor 488-NHS(0.4 μg/mL, degree of labeling 1.8). Nucleus were stained with Hoechist 33342. The cells were imaged using a fluorescence microscope.  Green indicates fluorescence of SaraFluor 488, blue indicates fluorescence of Hoechist 33342.

     

    A multicolor imaging example using SaraFluor 600-NHS and 700-NHS

    HeLa cells fixed and permeabilized with 1% Triton-X100 were reacted with rabbit anti-COX IV antibody (3E11, Cell Signaling Technology 1/500) and then, reacted with goat anti-rabbit antibody labeled with SaraFluor 600-NHS. In addition, cells were reacted with rat anti-tubulin antibody (YL 1/2, Novus Biologicals, 1/1000) and then, reacted with goat anti-rat antibody labeled with SaraFluor 700-NHS.  Nucleus were stained with Hoechist 33342. The cells were imaged using a fluorescence microscope.  Red indicates fluorescence of SaraFluor 600, Cyan indicated that of SaraFluor 700, blue indicates fluorescence of Hoechist 33342.

     

    A multicolor imaging example of SaraFluor 488-NHS and 650-NHS

    OVCAR5 cells fixed and permeabilized with 1% Triton-X100 were reacted with rabbit anti-α/β tubulin antibody (#2148, Cell Signaling Technology 1/50) and then, reacted with goat anti-rabbit antibody labeled using SaraFluor 488-NHS(0.4 μg/mL, degree of labeling 1.8). Nucleus were stained with Hoechist 33342. The cells were imaged using a fluorescence microscope.  Green indicates fluorescence of SaraFluor 488, blue indicates fluorescence of Hoechist 33342.

     

    A multicolor imaging example of SaraFluor 600-NHS and 700-NHS

     

    HeLa cells fixed and permeabilized with 1% Triton-X100 were reacted with rabbit anti-COX IV antibody (3E11, Cell Signaling Technology 1/500) and then, reacted with goat anti-rabbit antibody labeled with SaraFluor 600-NHS. In addition, cells were reacted with rat anti-tubulin antibody (YL 1/2, Novus Biologicals, 1/1000) and then, reacted with goat anti-rat antibody labeled with SaraFluor 700-NHS.  Nucleus were stained with Hoechist 33342. The cells were imaged using a fluorescence microscope.  Red indicates fluorescence of SaraFluor 600, Cyan indicated that of SaraFluor 700, blue indicates fluorescence of Hoechist 33342.

     

    An imaging example of SaraFluor 720-NHS

    HeLa cells fixed and permeabilized with 1% Triton-X100 were reacted with rat anti-tubulin antibody (YL 1/2, Novus Biologicals, 1/1000) and then, reacted with goat anti-rat antibody labeled using SaraFluor 720-NHS. The cells were imaged using a fluorescence microscope.

     

FAQ

  • Q How can I prepare 0.1 M sodium bicarbonate buffer? Why this buffer is required?
    A

    Dissolve sodium bicarbonate (NaHCO3) to pure water and adjust the concentration to be 0.1 M. The pH should be in the range of 8.0 to 8.4. Alternatively, you can adjust pH by adding a small amount of Na2CO3  or  HCl.

    The reason to recommend this buffer is that the reaction of NHS ester and primary amines is more efficient at alkaline pH. Instead, you can use other alkaline buffers such as HEPES/phosphate/borate buffers.  Avoid to use Tris buffer because it has primary amines and inhibit the reaction of NHS with the target molecules.

  • Q How can I get molar extinction coefficient of a protein at 280 nm?
    A

    Molar extinction coefficient of protein at 280 nm can be calculated by the methods of Gill and von Hippel (1989) Analytical Biochemistry, 182: 319-326  and Anthis and Clore (2013) Protein Science 22:851-858.

    You may find Web services to obtain them in the following sites. Goryo Chemical do not support the usage of these sites and use them according to the Terms and Conditions in each sites.

    http://protcalc.sourceforge.net/
    http://web.expasy.org/protparam/
    http://nickanthis.com/tools/a205.html

  • Q Tell me the efficient labeling method using NHS reagent.
    A

    Prepare purified antibody (or other proteins). If you intended to crude protein sample, we recommend to purify protein using either affinity column, ultrafilteration or gel filteration before the labeling to increase labeling efficiency. In addition, avoid to use Tris-buffer, because Tris has primary amine and it strongly inhibit the reaction of NHS to the target protein.

    In some cases, reaction at 4oC for overnight gives higher degree of labeling compared with reaction at 37oC for 1 hour.

  • Q What is the feature of SaraFluor labeling dye?
    A

    SaraFluor 488 and 600 are improved dye of fluorescein. SaraFluor 488 shows stable fluorescence intensity above pH 6, whereas fluorescein shows stable maximum fluorescence above pH 8. SaraFluor 600 shows stronger fluorescence than Texas Red. Unfortunately, it is not photostable enough compared to other major fluorophores, usage of antifade reagent is recommended in some cases.

    SaraFluor 650, 700, 720 are a silicone rhodamine (SiR) derived fluorophore. These show stable fluorescence irrespective to pH change. In addition, these are photostable fluorophores.

  • Q My question is not in this FAQ list.....
    A

    Please also refer the frequently asked questions on fluorescent probes

     

Reference

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