Actively helping customers, employees and the global community during the coronavirus SARS-CoV-2 outbreak.  Learn more >>

iFluor® 770 succinimidyl ester

Ordering information
Price ()
Catalog Number1369
Unit Size
Find Distributor
Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight1082.27
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.09
Correction Factor (280 nm)0.08
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)777
Emission (nm)797
Quantum yield0.16
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501
Alternative formats
iFluor® 770 maleimide

OverviewpdfSDSpdfProtocol


Molecular weight
1082.27
Correction Factor (260 nm)
0.09
Correction Factor (280 nm)
0.08
Extinction coefficient (cm -1 M -1)
2500001
Excitation (nm)
777
Emission (nm)
797
Quantum yield
0.16
AAT Bioquest's iFluor® dyes are optimized for labeling proteins, in particular, antibodies. These dyes are bright, photostable and have minimal quenching on proteins. They can be well excited by the major laser lines of fluorescence instruments (e.g., 350, 355, 405, 488, 555, 633, 638, 647, 660 and 802 nm). iFluor® 770 dyes have fluorescence excitation and emission maxima of ~770 nm and ~790 nm respectively. These spectral characteristics make them a unique color for fluorescence imaging and flow cytometry applications. iFluor® 770 is an excellent acceptor dye for preparing tandem colors with APC and PE. These iFluor® 770 tandem colors offer a set of unique color profiles for spectral flow cytometry. iFluor® 770 is one of the brightest NIR dyes, and some of its antibody conjugates are significantly brighter than the ones prepared from the IRDyes of the similar wavelengths such as IRDye800 RS. iFluor® 770 succinimidyl ester is amino-reactive form that can be used to conjugate with amino-containing molecules such as antibodies and peptides.

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. iFluor™ 770 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ 770 SE 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 iFluor™ 770 SE. You might need further optimization for your particular proteins. 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
  1. 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.
  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 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 iFluor® 770 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 mg0.5 mg1 mg5 mg10 mg
1 mM92.398 µL461.992 µL923.984 µL4.62 mL9.24 mL
5 mM18.48 µL92.398 µL184.797 µL923.984 µL1.848 mL
10 mM9.24 µL46.199 µL92.398 µL461.992 µL923.984 µL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.09
Correction Factor (280 nm)0.08
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)777
Emission (nm)797
Quantum yield0.16

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 350 succinimidyl ester3454502000010.9510.830.23
iFluor® 405 succinimidyl ester4034273700010.9110.480.77
iFluor® 430 succinimidyl ester4334984000010.7810.680.3
iFluor® 440 succinimidyl ester4344804000010.6710.3520.229
iFluor® 450 succinimidyl ester4515024000010.8210.450.27
iFluor® 460 succinimidyl ester468493800001~0.810.980.46
iFluor® 488 succinimidyl ester4915167500010.910.210.11
iFluor® 514 succinimidyl ester5115277500010.8310.2650.116
iFluor® 532 succinimidyl ester5375609000010.6810.260.16
iFluor® 546 succinimidyl ester54155710000010.6710.250.15
iFluor® 555 succinimidyl ester55757010000010.6410.230.14
iFluor® 560 succinimidyl ester56057112000010.5710.04820.069
iFluor® 568 succinimidyl ester56858710000010.5710.340.15
iFluor® 570 succinimidyl ester56057112000010.5810.0480.069
iFluor® 594 succinimidyl ester58860418000010.5310.050.04
iFluor® 597 succinimidyl ester59861810000010.710.3350.514
iFluor® 610 succinimidyl ester61062811000010.8510.320.49
iFluor® 633 succinimidyl ester64065425000010.2910.0620.044
iFluor® 647 succinimidyl ester65667025000010.2510.030.03
iFluor® 660 succinimidyl ester66367825000010.2610.070.08
iFluor® 665 succinimidyl ester667692110,00010.2210.120.09
iFluor® 670 succinimidyl ester67168220000010.5510.030.033
iFluor® 680 succinimidyl ester68470122000010.2310.0970.094
iFluor® 690 succinimidyl ester68570422000010.3010.090.06
iFluor® 700 succinimidyl ester69071322000010.2310.090.04
iFluor® 710 succinimidyl ester71773919000010.6010.120.07
iFluor® 720 succinimidyl ester71674024000010.1410.150.13
iFluor® 740 succinimidyl ester74276422500010.2010.160.16
iFluor® 750 succinimidyl ester75777927500010.1210.0440.039
iFluor® 780 succinimidyl ester78480825000010.1610.130.12
iFluor® 790 succinimidyl ester78781225000010.1310.10.09
iFluor® 800 succinimidyl ester80182025000010.1110.030.08
iFluor® 810 succinimidyl ester81182225000010.0510.090.15
iFluor® 820 succinimidyl ester8228502500001-0.110.16
iFluor® 830 succinimidyl ester830867----
iFluor® 840 succinimidyl ester8368792000001-0.20.09
iFluor® 860 succinimidyl ester8538782500001-0.10.14
Show More (38)

References


View all 50 references: Citation Explorer
Molecular imaging of a fluorescent antibody against epidermal growth factor receptor detects high-grade glioma.
Authors: Zhou, Quan and Vega Leonel, Johana C M and Santoso, Michelle Rai and Wilson, Christy and van den Berg, Nynke S and Chan, Carmel T and Aryal, Muna and Vogel, Hannes and Cayrol, Romain and Mandella, Michael J and Schonig, Frank and Lu, Guolan and Gambhir, Sanjiv S and Moseley, Michael E and Rosenthal, Eben L and Grant, Gerald A
Journal: Scientific reports (2021): 5710
Comparison of HER2-Targeted Antibodies for Fluorescence-Guided Surgery in Breast Cancer.
Authors: AghaAmiri, Solmaz and Simien, Jo and Thompson, Alastair M and Voss, Julie and Ghosh, Sukhen C and Hernandez Vargas, Servando and Kim, Sarah and Azhdarinia, Ali and Tran Cao, Hop S
Journal: Molecular imaging (2021): 5540569
Predicting Schwannoma Growth in a Tumor Model Using Targeted Imaging.
Authors: Morrison, Daniel R and Sorace, Anna G and Hamilton, Ellis and Moore, Lindsay S and Houson, Hailey A and Udayakumar, Neha and Ovaitt, Alyssa and Warram, Jason M and Walsh, Erika M
Journal: Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Aca (2021): e615-e623
Resection and survival data from a clinical trial of glioblastoma multiforme-specific IRDye800-BBN fluorescence-guided surgery.
Authors: He, Kunshan and Chi, Chongwei and Li, Deling and Zhang, Jingjing and Niu, Gang and Lv, Fangqiao and Wang, Junmei and Che, Wenqiang and Zhang, Liwei and Ji, Nan and Zhu, Zhaohui and Tian, Jie and Chen, Xiaoyuan
Journal: Bioengineering & translational medicine (2021): e10182
Preclinical Development of Near-Infrared-Labeled CD38-Targeted Daratumumab for Optical Imaging of CD38 in Multiple Myeloma.
Authors: Cho, Nicholas and Ko, Sooah and Shokeen, Monica
Journal: Molecular imaging and biology (2021): 186-195
Tissue biodistribution and tumor targeting of near-infrared labelled anti-CD38 antibody-drug conjugate in preclinical multiple myeloma.
Authors: Cho, Nicholas and Ko, Sooah and Shokeen, Monica
Journal: Oncotarget (2021): 2039-2050
EGFR-targeted intraoperative fluorescence imaging detects high-grade glioma with panitumumab-IRDye800 in a phase 1 clinical trial.
Authors: Zhou, Quan and van den Berg, Nynke S and Rosenthal, Eben L and Iv, Michael and Zhang, Michael and Vega Leonel, Johana C M and Walters, Shannon and Nishio, Naoki and Granucci, Monica and Raymundo, Roan and Yi, Grace and Vogel, Hannes and Cayrol, Romain and Lee, Yu-Jin and Lu, Guolan and Hom, Marisa and Kang, Wenying and Hayden Gephart, Melanie and Recht, Larry and Nagpal, Seema and Thomas, Reena and Patel, Chirag and Grant, Gerald A and Li, Gordon
Journal: Theranostics (2021): 7130-7143
Photoacoustic Molecular Imaging for the Identification of Lymph Node Metastasis in Head and Neck Cancer Using an Anti-EGFR Antibody-Dye Conjugate.
Authors: Nishio, Naoki and van den Berg, Nynke S and Martin, Brock A and van Keulen, Stan and Fakurnejad, Shayan and Rosenthal, Eben L and Wilson, Katheryne E
Journal: Journal of nuclear medicine : official publication, Society of Nuclear Medicine (2021): 648-655
Ferritin nanocages for early theranostics of tumors via inflammation-enhanced active targeting.
Authors: Jiang, Bing and Jia, Xiaohua and Ji, Tianjiao and Zhou, Meng and He, Jiuyang and Wang, Kun and Tian, Jie and Yan, Xiyun and Fan, Kelong
Journal: Science China. Life sciences (2021)
Multi-Modal Imaging Probe for Glypican-3 Overexpressed in Orthotopic Hepatocellular Carcinoma.
Authors: Feng, Shuo and Meng, Xiaoqing and Li, Zhao and Chang, Tse-Shao and Wu, Xiaoli and Zhou, Juan and Joshi, Bishnu and Choi, Eun-Young and Zhao, Lili and Zhu, Jiye and Wang, Thomas D
Journal: Journal of medicinal chemistry (2021): 15639-15650