ATTO 495 NHS ester
ATTO 495 is a green fluorescent dye derived from acridine orange, known for its strong absorption, high fluorescence quantum yield, excellent photostability, and superior thermal stability. It exhibits moderate hydrophilicity and is highly soluble in polar solvents such as DMF and DMSO, with an optimal excitation range of 465-510 nm. Notably, ATTO 495 exhibits intense and long-lived phosphorescence in solid matrices or at low temperatures. This dye is well-suited for advanced applications in single-molecule detection and high-resolution microscopy techniques, such as PALM, dSTORM, and STED microscopy. It is also compatible with flow cytometry (FACS), fluorescence in situ hybridization (FISH), and a wide range of other biological assays.
The N-hydroxysuccinimidyl (NHS) ester of ATTO 495 is a widely used reagent for the conjugation of this dye to proteins or antibodies. NHS esters react selectively and efficiently with primary amines (such as the side chains of lysine residues or aminosilane-coated surfaces) at pH 7-9, forming stable covalent amide bonds. This property makes ATTO 495 NHS ester an excellent choice for labeling proteins, amine-modified oligonucleotides, and other amine-containing molecules.
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
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.
Add anhydrous DMSO into the vial of ATTO 495 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 495 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.
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.
Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes.
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 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.
The Degree of Substitution (DOS) is the most important factor for characterizing dye-labeled protein. Proteins of lower DOS usually have weaker fluorescence intensity, but proteins of higher DOS (e.g., DOS > 6) tend to have reduced fluorescence too. The optimal DOS for most antibodies is recommended between 2 and 10, depending on the properties of dye and protein. For effective labeling, the degree of substitution should be controlled to have 6-8 moles of ATTO 495 NHS ester to one mole of antibody. The following steps are used to determine the DOS of ATTO 495 NHS ester-labeled proteins.
To measure the absorption spectrum of a dye-protein conjugate, it is recommended to keep the sample concentration in the range of 1-10 µM depending on the extinction coefficient of the dye.
For most spectrophotometers, the sample (from the column fractions) needs to be diluted with de-ionized water so that the O.D. values are in the range of 0.1 to 0.9. The O.D. (absorbance) at 280 nm is the maximum absorption of protein, while 525 nm is the maximum absorption of ATTO 495 NHS ester. To obtain accurate DOS, ensure the conjugate is free of the non-conjugated dye.
You can calculate the DOS using our tool by following this link:
Spectrum
Product family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
ATTO 488 NHS ester | 499 | 520 | 90000 | 0.80 | 0.22 | 0.09 |
ATTO 532 NHS ester | 531 | 552 | 115000 | 0.90 | 0.22 | 0.11 |
ATTO 647 NHS ester | 646 | 666 | 120000 | 0.20 | 0.08 | 0.04 |
ATTO 647N NHS ester | 645 | 663 | 150000 | 0.651 | 0.06 | 0.05 |
ATTO 594 NHS ester | 602 | 621 | 120000 | 0.85 | 0.26 | 0.51 |
ATTO 514 NHS ester | 510 | 531 | 115,000 | 0.85 | 0.21 | 0.08 |
ATTO 565 NHS ester | 562 | 589 | 120000 | 0.90 | 0.27 | 0.12 |
ATTO 390 NHS ester | 390 | 475 | 24000 | .90 | 0.46 | 0.09 |
ATTO 425 NHS ester | 438 | 484 | 45000 | 0.90 | 0.19 | 0.17 |
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References
Authors: Rode, Sascha and Hayn, Manuel and Röcker, Annika and Sieste, Stefanie and Lamla, Markus and Markx, Daniel and Meier, Christoph and Kirchhoff, Frank and Walther, Paul and Fändrich, Marcus and Weil, Tanja and Münch, Jan
Journal: Bioconjugate chemistry (2017): 1260-1270
Authors: Wang, Yi-Rou and Yang, Yuan-Han and Lu, Chi-Yu and Chen, Su-Hwei
Journal: Analytica chimica acta (2015): 76-82