zFluor™ 633 succinimidyl ester

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	1163.33
Solvent	DMSO

Spectral properties

Excitation (nm)	623
Emission (nm)	639

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501

Overview SDS Protocol

Molecular weight

1163.33

Excitation (nm)

623

Emission (nm)

639

Our zFluor™ serial dyes are developed to have the possibly highest photostability at a given wavelength compared to the other similar wavelength dyes on the market. zFluor™ 633 has almost identical spectral properties to ATTO 633, ATTO 635 and Alexa Fluor® 633 (ThermoFisher). Its high thermal and photostability makes it an excellent choice for single-molecule detection applications and high-resolution microscopy such as PALM, dSTORM and STED. Under the same test conditions, zFluor™ 633 has much higher ozone stability, making it a much better choice for microarray and other biochip-based applications. In addition, zFluor™-labeled oligonucleotides and peptides are much brighter and more photostable than the ones labeled by Alexa Fluor® 647 and Cy5®. This feature is extremely useful for fluorescence in-situ hybridization (FISH). In common with our iFluor® labels, the absorption and fluorescence of our zFluor™ 633 are independent of pH in the range of pH 2 to 11. It is well excited at 633 nm of He-Ne laser, the 633 nm line of the He-Ne laser.

Example protocol

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Protein stock solution (Solution A)

Mix 100 µL of a reaction buffer (e.g., 1 M sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) with 900 µL of the target protein solution (e.g. antibody, protein concentration >2 mg/mL if possible) to give 1 mL protein labeling stock solution. Note: The pH of the protein solution (Solution A) should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer. Note: The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation. Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results. Note: The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.

2. zFluor™ 633 succinimidyl ester stock solution (Solution B)

Add anhydrous DMSO into the vial of zFluor™ 633 succinimidyl ester to make a 10 mM stock solution. Mix well by pipetting or vortex. Note: Prepare the dye stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with zFluor™ 633 succinimidyl ester. You might need further optimization for your particular proteins. Note: Each protein requires distinct dye/protein ratio, which also depends on the properties of dyes. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low dye/protein ratio gives reduced sensitivity.

Run conjugation reaction

Use 10:1 molar ratio of Solution B (dye)/Solution A (protein) as the starting point: Add 5 µL of the dye stock solution (Solution B, assuming the dye stock solution is 10 mM) into the vial of the protein solution (95 µL of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD. Note: We recommend to use 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too less or too high, determine the optimal dye/protein ratio at 5:1, 15:1 and 20:1 respectively.
Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes.

Purify the conjugation

The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.

Prepare Sephadex G-25 column according to the manufacture instruction.
Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate. Note: For immediate use, the dye-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses. Note: For longer term storage, dye-protein conjugate solution need be concentrated or freeze dried.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of zFluor™ 633 succinimidyl ester to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

	0.1 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	85.96 µL	429.801 µL	859.601 µL	4.298 mL	8.596 mL
5 mM	17.192 µL	85.96 µL	171.92 µL	859.601 µL	1.719 mL
10 mM	8.596 µL	42.98 µL	85.96 µL	429.801 µL	859.601 µL

Molarity calculator

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Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
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Spectrum

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Spectral properties

Excitation (nm)	623
Emission (nm)	639

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
zFluor™ 647 succinimidyl ester	648	668	-	-	-	-
zFluor™ 635 succinimidyl ester	-	-	110,000¹	0.22¹	0.12	0.09
iFluor® 633 succinimidyl ester	640	654	250000¹	0.29¹	0.062	0.044

Images

Figure 1. Fluorescent dye NHS esters (or succinimidyl esters) are the most popular tool for conjugating dyes to a peptide, protein, antibody, amino-modified oligonucleotide or nucleic acid. NHS esters react readily with the primary amines (R-NH₂) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting dye conjugates are quite stable.

References

View all 19 references: Citation Explorer

Single nucleotide polymorphism discrimination with and without an ethidium bromide intercalator
Authors: Fenati, R. A.; Connolly, A. R.; Ellis, A. V.
Journal: Anal Chim Acta (2017): 121-128

Generation and Characterization of Virus-Enhancing Peptide Nanofibrils Functionalized with Fluorescent Labels
Authors: Rode, S.; Hayn, M.; Rocker, A.; Sieste, S.; Lamla, M.; Markx, D.; Meier, C.; Kirchhoff, F.; Walther, P.; F and rich, M.; Weil, T.; Munch, J.
Journal: Bioconjug Chem (2017): 1260-1270

Capillary-Driven Microfluidic Chips for Miniaturized Immunoassays: Patterning Capture Antibodies Using Microcontact Printing and Dry-Film Resists
Authors: Temiz, Y.; Lovchik, R. D.; Delamarche, E.
Journal: Methods Mol Biol (2017): 37-47

Background Suppression in Imaging Gold Nanorods through Detection of Anti-Stokes Emission
Authors: Carattino, A.; Keizer, V. I.; Schaaf, M. J.; Orrit, M.
Journal: Biophys J (2016): 2492-2499

Absolute two-photon excitation spectra of red and far-red fluorescent probes
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Journal: Opt Lett (2015): 4915-8

Diffusion coefficients and dissociation constants of enhanced green fluorescent protein binding to free standing membranes
Authors: Thomas, F. A.; Visco, I.; Petrasek, Z.; Heinemann, F.; Schwille, P.
Journal: Data Brief (2015): 537-41

Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy
Authors: Wu, Y.; Wu, X.; Lu, R.; Zhang, J.; Toro, L.; Stefani, E.
Journal: Sci Rep (2015): 14766

The contribution of reactive oxygen species to the photobleaching of organic fluorophores
Authors: Zheng, Q.; Jockusch, S.; Zhou, Z.; Blanchard, S. C.
Journal: Photochem Photobiol (2014): 448-454

Topical drug delivery to retinal pigment epithelium with microfluidizer produced small liposomes
Authors: Lajunen, T.; Hisazumi, K.; Kanazawa, T.; Okada, H.; Seta, Y.; Yliperttula, M.; Urtti, A.; Takashima, Y.
Journal: Eur J Pharm Sci (2014): 23-32

Ultrasensitive detection of lead (II) based on fluorescent aptamer-functionalized carbon nanotubes
Authors: Taghdisi, S. M.; Emrani, S. S.; Tabrizian, K.; Ramezani, M.; Abnous, K.; Emrani, A. S.
Journal: Environ Toxicol Pharmacol (2014): 1236-42