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”.

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Products

Code No.	Product Name	Size
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

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Product Information

SaraFluor™ 650
Product Information

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Properties of SaraFluor series

Product name	Abs_max (nm)	FL_max (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 4^oC for overnight gives higher degree of labeling compared with reaction at 37^oC 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