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ATTO 532 NHS ester

Fluorescent ATTO dye NHS esters (or succinimidyl esters) are the most popular tool for conjugating ATTO dyes to a peptide, protein, antibody, amino-modified oligonucleotide or nucleic acid. NHS esters react readily with the primary amines (R-NH<sub>2</sub>) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting dye conjugates are quite stable.
Fluorescent ATTO dye NHS esters (or succinimidyl esters) are the most popular tool for conjugating ATTO dyes to a peptide, protein, antibody, amino-modified oligonucleotide or nucleic acid. NHS esters react readily with the primary amines (R-NH<sub>2</sub>) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting dye conjugates are quite stable.
Fluorescent ATTO dye NHS esters (or succinimidyl esters) are the most popular tool for conjugating ATTO dyes to a peptide, protein, antibody, amino-modified oligonucleotide or nucleic acid. NHS esters react readily with the primary amines (R-NH<sub>2</sub>) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting dye conjugates are quite stable.
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Physical properties
Molecular weight843.96
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.22
Correction Factor (280 nm)0.11
Extinction coefficient (cm -1 M -1)115000
Excitation (nm)531
Emission (nm)552
Quantum yield0.90
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Molecular weight
843.96
Correction Factor (260 nm)
0.22
Correction Factor (280 nm)
0.11
Extinction coefficient (cm -1 M -1)
115000
Excitation (nm)
531
Emission (nm)
552
Quantum yield
0.90
ATTO 532 is a rhodamine-based fluorescent labeling dye with high molecular high extinction coefficient and fluorescence quantum yield (0.90) as well as sufficient Stoke's shift (excitation maximum 532 nm, emission maximum 552 nm). The dye also has relatively good photostability. It is optimized for excitation with frequency doubled Nd:YAG-Laser. ATTO 532 NHS ester is used for labeling amino-containing molecules such as proteins, peptides and amino-modified oligonucleotides.

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

Protein stock solution (Solution A)
  1. 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. The final protein concentration range of 2-10 mg/mL is recommended for optimal labeling efficiency.

ATTO 532 NHS ester stock solution (Solution B)
  1. Add anhydrous DMSO into the vial of ATTO 532 NHS 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 the 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 ATTO 532 NHS ester. You might need further optimization for your particular proteins.

Note: Each protein requires a distinct dye/protein ratio, which also depends on the properties of dyes. Over-labeling of a protein could detrimentally affect its binding affinity, while the protein conjugates of low dye/protein ratio give reduced sensitivity.

Run conjugation reaction
  1. Use a 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 using a 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.

  2. 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.

  1. Prepare Sephadex G-25 column according to the manufacture instruction.
  2. Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
  3. Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
  4. 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 must be diluted with staining buffer, and aliquoted for multiple uses.

    Note: For longer-term storage, the dye-protein conjugate solution needs to be concentrated or freeze-dried.

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of ATTO 532 NHS 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 mg0.5 mg1 mg5 mg10 mg
1 mM118.489 µL592.445 µL1.185 mL5.924 mL11.849 mL
5 mM23.698 µL118.489 µL236.978 µL1.185 mL2.37 mL
10 mM11.849 µL59.245 µL118.489 µL592.445 µL1.185 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


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spectrum

Spectral properties

Correction Factor (260 nm)0.22
Correction Factor (280 nm)0.11
Extinction coefficient (cm -1 M -1)115000
Excitation (nm)531
Emission (nm)552
Quantum yield0.90

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
ATTO 488 NHS ester499520900000.800.250.10
ATTO 647 NHS ester6466661200000.200.080.04
ATTO 647N NHS ester6456631500000.6510.060.05
ATTO 594 NHS ester6026211200000.850.260.51
ATTO 514 NHS ester510531115,0000.850.210.08

Images


Citations


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Authors: Wang, J., Zhou, Z., Zhang, F., Xu, H., Chen, W., Jiang, T.
Journal: Colloids Surf B Biointerfaces (2018): 303-311
Cell-permeable organic fluorescent probes for live-cell long-term super-resolution imaging reveal lysosome-mitochondrion interactions
Authors: Han, Y., Li, M., Qiu, F., Zhang, M., Zhang, Y. H.
Journal: Nat Commun (2017): 1307
Field-Controlled Charge Separation in a Conductive Matrix at the Single-Molecule Level: Toward Controlling Single-Molecule Fluorescence Intermittency
Authors: Kennes, K., Dedecker, P., Hutchison, J. A., Fron, E., Uji, I. H., Hofkens, J., Van der Auweraer, M.
Journal: ACS Omega (2016): 1383-1392
Determination of equilibrium and rate constants for complex formation by fluorescence correlation spectroscopy supplemented by dynamic light scattering and Taylor dispersion analysis
Authors: Zhang, X., Poniewierski, A., Jelinska, A., Zagozdzon, A., Wisniewska, A., Hou, S., Holyst, R.
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A Cystine Knot Peptide Targeting Integrin alphavbeta6 for Photoacoustic and Fluorescence Imaging of Tumors in Living Subjects
Authors: Zhang, C., Kimura, R., Abou-Elkacem, L., Levi, J., Xu, L., Gambhir, S. S.
Journal: J Nucl Med (2016): 1629-1634
Tracking structural transitions of bovine serum albumin in surfactant solutions by fluorescence correlation spectroscopy and fluorescence lifetime analysis
Authors: Zhang, X., Poniewierski, A., Hou, S., Sozanski, K., Wisniewska, A., Wieczorek, S. A., Kalwarczyk, T., Sun, L., Holyst, R.
Journal: Soft Matter (2015): 2512-8
The effect of local dynamics of Atto 390-labeled lysozyme on fluorescence anisotropy modeling
Authors: Babcock, J. J., Brancaleon, L.
Journal: Biopolymers (2015): 285-95
Application of single molecule fluorescence microscopy to characterize the penetration of a large amphiphilic molecule in the stratum corneum of human skin
Authors: Volz, P., Boreham, A., Wolf, A., Kim, T. Y., Balke, J., Frombach, J., Hadam, S., Afraz, Z., Rancan, F., Blume-Peytavi, U., Vogt, A., Alexiev, U.
Journal: Int J Mol Sci (2015): 6960-77
AMBER-DYES: Characterization of Charge Fluctuations and Force Field Parameterization of Fluorescent Dyes for Molecular Dynamics Simulations
Authors: Graen, T., Hoefling, M., Grubmuller, H.
Journal: J Chem Theory Comput (2014): 5505-12
The key role of geminate electron-hole pair recombination in the delayed fluorescence in rhodamine 6G and ATTO-532
Authors: Aydemir, M., Jankus, V., Dias, F. B., Monkman, A.
Journal: Phys Chem Chem Phys (2014): 21543-9

References


View all 1 references: Citation Explorer
Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates
Authors: Berlier JE, Rothe A, Buller G, Bradford J, Gray DR, Filanoski BJ, Telford WG, Yue S, Liu J, Cheung CY, Chang W, Hirsch JD, Beechem JM, Haugl and RP., undefined
Journal: J Histochem Cytochem (2003): 1699