XFD790 NHS Ester
Product key features
- Ex/Em: 782/805 nm
- Extinction coefficient: 260,000 cm-1M-1
- Reactive Group: NHS ester
- Easy Conjugation: Efficiently labels primary amines on proteins, ligands, and amine-modified oligonucleotides
- Water Soluble: Minimizes aggregation, enhancing signal clarity for advanced imaging and live-cell studies
- Reduced Autofluorescence: Long-wavelength emission minimizes background interference, enhancing signal-to-noise ratios in complex biological matrices.
- Panel Optimization: Well-separated from far-red fluorophores such as iFluor® 647 and allophycocyanin (APC), facilitating precise multicolor fluorescence detection.
Product description
XFD790, manufactured by AAT Bioquest, is a highly efficient near-infrared fluorescent dye that is structurally identical to Alexa Fluor™ 790 (ThermoFisher). Spectrally analogous to indocyanine green (ICG) and IRDye™ 800, XFD790 demonstrates exceptional aqueous solubility and sustained fluorescence stability over a broad pH range (pH 4–10), ensuring consistent and reproducible performance across diverse experimental conditions. Its long-wavelength emission effectively mitigates background autofluorescence, thereby enhancing signal-to-noise ratios in complex biological matrices, including tissue samples. As the longest-wavelength fluorophore in the XFD series, XFD790 offers superior spectral separation from widely used far-red fluorophores such as iFluor® 647, XFD647, and allophycocyanin (APC), facilitating precise multicolor fluorescence analyses. Furthermore, its optical properties make it an excellent candidate for small animal in vivo imaging (SAIVI) and two-color western blot applications using the LI-COR™ Odyssey™ infrared imaging system.
The N-hydroxysuccinimidyl (NHS) ester of XFD790 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 XFD790 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
Prepare a 1 mL protein labeling stock solution by mixing 100 µL of reaction buffer (such as 1 M sodium carbonate solution or 1 M phosphate buffer, pH ~9.0) with 900 µL of the target protein solution (e.g., an antibody with a protein concentration of at least 2 mg/mL, if possible).
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 it to within the 8.0-9.0 range using either 1 M sodium bicarbonate solution or 1 M phosphate buffer at pH 9.0.
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, dialyze it against 1X PBS, pH 7.2-7.4, to remove any free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) commonly used in protein precipitation.
Note: Antibodies that are impure or stabilized with bovine serum albumin (BSA) or gelatin may not label effectively. Additionally, sodium azide or thimerosal can interfere with the conjugation reaction. To achieve optimal labeling results, these preservatives should be removed through dialysis or spin column techniques.
Note: For optimal labeling efficiency, it is recommended to maintain a final protein concentration between 2-10 mg/mL. Protein concentrations below 2 mg/mL can significantly reduce conjugation efficiency.
To prepare a 10 mM stock solution of XFD790 NHS ester, add anhydrous DMSO directly to the vial of XFD790 NHS ester. Mix well by pipetting or vortexing.
Note: Prepare the dye stock solution (Solution B) before starting the conjugation, and use it promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in the freezer for up to two weeks, provided it is protected from light and moisture. Avoid freeze-thaw cycles.
SAMPLE EXPERIMENTAL PROTOCOL
This protocol is designed for labeling Goat anti-mouse IgG with XFD790 NHS ester. Additional optimization may be required to adapt the protocol to your specific proteins.
Note: Each protein requires a distinct dye/protein ratio, which varies depending on the characteristics of the dye. Over-labeling a protein can negatively impact its binding affinity, whereas using a low dye-to-protein ratio in protein conjugates can result in reduced sensitivity.
Use a 10:1 molar ratio of Solution B (dye) to 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) to the vial containing 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 low 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 demonstrates the purification of a dye-protein conjugate using a Sephadex G-25 column.
Prepare the Sephadex G-25 column according to the manufacturer's instructions.
Carefully load the reaction mixture (from the "Run Conjugation Reaction" step) 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 a critical factor in characterizing dye-labeled proteins. Proteins with a lower DOS generally exhibit weaker fluorescence, while those with a higher DOS (e.g., DOS > 6) may also show reduced fluorescence. The optimal DOS for most antibodies typically ranges between 2 and 10, depending on the specific properties of both the dye and the protein. For effective labeling, it is recommended to achieve a DOS of 6-8 moles of XFD790 NHS ester per mole of antibody. The following steps outline the process for determining the DOS of XFD790 NHS ester-labeled proteins.
For accurate measurement of the absorption spectrum of a dye-protein conjugate, maintain the sample concentration between 1-10 µM, adjusting as needed based on the dye's extinction coefficient.
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 782 nm is the maximum absorption of XFD790 NHS ester. To obtain accurate DOS, ensure the conjugate is free of the non-conjugated dye.
You can calculate DOS using our tool by following this link:
Spectrum
Product family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Correction Factor (260 nm) | Correction Factor (280 nm) |
XFD488 NHS Ester *Same Structure to Alexa Fluor™ 488 NHS Ester* | 499 | 520 | 71000 | 0.30 | 0.11 |
XFD350 NHS Ester *Same Structure to Alexa Fluor™ 350 NHS Ester* | 343 | 441 | 19000 | 0.25 | 0.19 |
XFD532 NHS Ester *Same Structure to Alexa Fluor™ 532 NHS Ester* | 534 | 553 | 81000 | 0.24 | 0.09 |
XFD594 NHS Ester *Same Structure to Alexa Fluor™ 594 NHS Ester* | 590 | 618 | 90000 | 0.43 | 0.56 |
XFD555 NHS Ester *Same Structure to Alexa Fluor™ 555 NHS Ester* | 553 | 568 | 150000 | 0.08 | 0.08 |
XFD647 NHS Ester *Same Structure to Alexa Fluor™ 647 NHS Ester* | 650 | 671 | 239000 | 0.00 | 0.03 |
XFD680 NHS Ester *Same Structure to Alexa Fluor™ 680 NHS Ester* | 681 | 704 | 184000 | 0.00 | 0.05 |
XFD700 NHS Ester *Same Structure to Alexa Fluor™ 700 NHS Ester* | 696 | 719 | 192000 | 0.00 | 0.07 |
XFD750 NHS Ester *Same Structure to Alexa Fluor™ 750 NHS Ester* | 752 | 776 | 240000 | 0.00 | 0.04 |
Show More (18) |
References
Authors: Manoharan, Gririraj and Bösel, Petra and Thien, Jannis and Holtmannspötter, Michael and Meingast, Laura and Schmidt, Mercedes and Eickmeier, Henning and Haase, Markus and Maultzsch, Janina and Steinhart, Martin and Wollschläger, Joachim and Palma, Matteo and Meyer, Carola
Journal: Molecules (Basel, Switzerland) (2023)
Authors: Oyarzun Mejia, Alejandra P and Hyman, Michael R
Journal: FEMS microbiology letters (2023)
Authors: Yan, Pengyu and Chen, Dan and Yan, Xutao and Yan, Xiaoting and Wang, Yingpeng and Liu, Chao and Yang, Xiaofeng
Journal: Frontiers in oncology (2022): 825476
Authors: Yang, Yongjun and Yan, Xiaoting and Li, Jiawei and Liu, Chao and Yang, Xiaofeng
Journal: Molecular therapy oncolytics (2022): 319-330
Authors: Pape, Luca Julius and Hambach, Julia and Gebhardt, Anna Josephine and Rissiek, Björn and Stähler, Tobias and Tode, Natalie and Khan, Cerusch and Weisel, Katja and Adam, Gerhard and Koch-Nolte, Friedrich and Bannas, Peter
Journal: Frontiers in immunology (2022): 1010270