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AAT Bioquest

iFluor® 675 succinimidyl ester

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, 405, 488, 514, 555, 633, 647 and 808 nm). iFluor® 675 dyes have fluorescence excitation and emission maxima of ~677 nm and ~710 nm respectively. These spectral characteristics make iFluor® 675 dye a unique acceptor for preparing PE, APC and PerCP tandems that are widely used in flow cytometry applications, in particular, for spectral flow cytometry. iFluor® 675 dyes have excellent photostability. iFluor® 675 SE is stable and shows good reactivity and selectivity with proteins (such as antibodies).

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.

iFluor™ 675 SE stock solution (Solution B)
  1. Add anhydrous DMSO into the vial of iFluor™ 675 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 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 iFluor™ 675 SE. 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.

Spectrum

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® 488 succinimidyl ester4915167500010.910.210.11
iFluor® 514 succinimidyl ester5115277500010.8310.2650.116
iFluor® 532 succinimidyl ester5375609000010.6810.260.16
iFluor® 555 succinimidyl ester55757010000010.6410.230.14
iFluor® 594 succinimidyl ester58760320000010.5310.050.04
iFluor® 633 succinimidyl ester64065425000010.2910.0620.044
iFluor® 647 succinimidyl ester65667025000010.2510.030.03
iFluor® 660 succinimidyl ester66367825000010.2610.070.08
iFluor® 680 succinimidyl ester68470122000010.2310.0970.094
iFluor® 700 succinimidyl ester69071322000010.2310.090.04
iFluor® 750 succinimidyl ester75777927500010.1210.0440.039
iFluor® 610 succinimidyl ester61062811000010.8510.320.49
iFluor® 710 succinimidyl ester71673915000010.6010.120.07
iFluor® 790 succinimidyl ester78781225000010.1310.10.09
iFluor® 800 succinimidyl ester80182025000010.1110.030.08
iFluor® 810 succinimidyl ester81182225000010.0510.090.15
iFluor® 820 succinimidyl ester82285025000010.110.16
iFluor® 860 succinimidyl ester85387825000010.10.14
iFluor® 546 succinimidyl ester54155710000010.6710.250.15
iFluor® 568 succinimidyl ester56858710000010.5710.340.15
iFluor® 430 succinimidyl ester4334984000010.7810.680.3
iFluor® 450 succinimidyl ester4515024000010.8210.450.27
iFluor® 840 succinimidyl ester8368792000001-0.20.09
iFluor® 560 succinimidyl ester56057112000010.5710.04820.069
iFluor® 670 succinimidyl ester67168220000010.5510.030.033
iFluor® 460 succinimidyl ester468493800001~0.810.980.46
iFluor® 440 succinimidyl ester4344804000010.6710.3520.229
iFluor® 665 succinimidyl ester667692110,00010.2210.120.09
iFluor® 690 succinimidyl ester68570422000010.3010.090.06
iFluor® 720 succinimidyl ester71674024000010.1410.150.13
iFluor® 740 succinimidyl ester74076422500010.2010.160.16
iFluor® 597 succinimidyl ester59861810000010.710.3350.514
iFluor® 770 succinimidyl ester77779725000010.160.090.08
iFluor® 780 succinimidyl ester78480825000010.1610.130.12
iFluor® 570 succinimidyl ester55757012000010.581--
iFluor® 830 succinimidyl ester830867----
iFluor® 620 succinimidyl ester621636---0.04
iFluor® 605 succinimidyl ester603623----
iFluor® 625 succinimidyl ester624640----
iFluor® 510 succinimidyl ester511530----
iFluor® 540 succinimidyl ester540557---0.105
iFluor® 445 succinimidyl ester446558----
iFluor® 500 succinimidyl ester501520----
Show More (36)

References

View all 37 references: Citation Explorer
A decade of blood-brain barrier permeability assays: Revisiting old traumatic brain injury rat data for new insights and experimental design.
Authors: Bolden, Chris T and Olson, Scott D and Cox, Charles S
Journal: Microvascular research (2023): 104453
A novel anti-TNF-α drug ozoralizumab rapidly distributes to inflamed joint tissues in a mouse model of collagen induced arthritis.
Authors: Oyama, Shohei and Ebina, Kosuke and Etani, Yuki and Hirao, Makoto and Kyuuma, Masanao and Fujii, Yasuyuki and Iwata, Katsuya and Ogawa, Bunichiro and Hasegawa, Tomoya and Kawano, Sasagu and Nakanishi, Yutaka and Okada, Seiji and Nakata, Ken
Journal: Scientific reports (2022): 18102
CD38-specific nanobodies allow in vivo imaging of multiple myeloma under daratumumab therapy.
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
A 15-min non-competitive homogeneous assay for microcystin and nodularin based on time-resolved Förster resonance energy transfer (TR-FRET).
Authors: Akter, Sultana and Lamminmäki, Urpo
Journal: Analytical and bioanalytical chemistry (2021): 6159-6170
Alpha-MSH Targeted Liposomal Nanoparticle for Imaging in Inflammatory Bowel Disease (IBD).
Authors: Peñate-Medina, Tuula and Damoah, Christabel and Benezra, Miriam and Will, Olga and Kairemo, Kalevi and Humbert, Jana and Sebens, Susanne and Peñate-Medina, Oula
Journal: Current pharmaceutical design (2020): 3840-3846
Page updated on October 8, 2024

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Catalog Number1043
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Physical properties

Molecular weight

1146.18

Solvent

DMSO

Spectral properties

Correction Factor (280 nm)

0.066

Excitation (nm)

683

Emission (nm)

700

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
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