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iFluor® 625 succinimidyl ester

iFluor® 625 succinimidyl ester is the most convenient reactive form of iFluor® 625 dyes that can be readily used for labeling proteins such as antibodies. It is particularly useful for preparing PE tandems, a new unique color for multiplex flow cytometric applications. iFluor®625 is a fluorophore from the iFluor® family, which is known for its bright red fluorescence and compatibility with various fluorescence techniques and instruments. When excited with light in the red range (around 600 to 650 nm), iFluor® 625 emits strong red fluorescence. It can also be well excited with red lasers at 633 and 647 nm. iFluor® 625 can be conjugated to a variety of biomolecules, including antibodies, proteins, nucleic acids, and small molecules, enabling their visualization and tracking in cells and tissues. It is commonly used in fluorescence microscopy, immunohistochemistry, flow cytometry, and other fluorescence-based assays. The iFluor® dyes, including iFluor® 625, are designed to exhibit high brightness, photostability, and minimal background noise, making them ideal for demanding imaging applications. They are also compatible with multiplexing experiments, allowing for simultaneous visualization of multiple targets using different iFluor® dyes with distinct emission wavelengths.

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 bicarbonate solution or 1 M phosphate buffer with pH ~8.5 to 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® 625 SE stock solution (Solution B)
  1. Add anhydrous DMSO into the vial of iFluor® 625 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® 625 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.

Characterize the Desired Dye-Protein conjugate

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 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 5-8 moles of iFluor® 625 SE to one mole of antibody. The following steps are used to determine the DOS of iFluor® 625 SE-labeled proteins.

Measure Absorption

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.

Read OD (absorbance) at 280 nm and dye maximum absorption (ƛmax = 624 nm for iFluor® 625 dyes)

For most spectrophotometers, the sample (from the column fractions) needs to be diluted with de-ionized water so that the OD 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 624 nm is the maximum absorption of iFluor® 625 SE. To obtain accurate DOS, make sure that the conjugate is free of the non-conjugated dye.

Calculate DOS

You can calculate DOS using our tool by following this link:

https://www.aatbio.com/tools/degree-of-labeling-calculator

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® 675 succinimidyl ester683700---0.066
iFluor® 620 succinimidyl ester621636---0.04
iFluor® 605 succinimidyl ester603623----
iFluor® 510 succinimidyl ester511530----
iFluor® 540 succinimidyl ester540557---0.105
iFluor® 445 succinimidyl ester446558----
iFluor® 500 succinimidyl ester501520800001-0.2060.088
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References

View all 17 references: Citation Explorer
Ex-Vivo Adhesion of Enterococcus faecalis and Enterococcus faecium to the Intestinal Mucosa of Healthy Beagles.
Authors: Hanifeh, Mohsen and Spillmann, Thomas and Huhtinen, Mirja and Sclivagnotis, Yannes S and Grönthal, Thomas and Hynönen, Ulla
Journal: Animals : an open access journal from MDPI (2021)
Analysis of lung stromal expression of the atypical chemokine receptor ACKR2 reveals unanticipated expression in murine blood endothelial cells.
Authors: Hansell, Christopher A H and Love, Samantha and Pingen, Marieke and Wilson, Gillian J and MacLeod, Megan and Graham, Gerard J
Journal: European journal of immunology (2020): 666-675
Inhibition of HMGB1/RAGE-mediated endocytosis by HMGB1 antagonist box A, anti-HMGB1 antibodies, and cholinergic agonists suppresses inflammation.
Authors: Yang, Huan and Liu, Hui and Zeng, Qiong and Imperato, Gavin H and Addorisio, Meghan E and Li, Jianhua and He, Mingzhu and Cheng, Kai Fan and Al-Abed, Yousef and Harris, Helena E and Chavan, Sangeeta S and Andersson, Ulf and Tracey, Kevin J
Journal: Molecular medicine (Cambridge, Mass.) (2019): 13
Epidermal growth factor receptor-targeted molecular imaging of colorectal tumors: Detection and treatment evaluation of tumors in animal models.
Authors: Miyamoto, Yoshihiko and Muguruma, Naoki and Fujimoto, Shota and Okada, Yasuyuki and Kida, Yoshifumi and Nakamura, Fumika and Tanaka, Kumiko and Nakagawa, Tadahiko and Kitamura, Shinji and Okamoto, Koichi and Miyamoto, Hiroshi and Sato, Yasushi and Takayama, Tetsuji
Journal: Cancer science (2019): 1921-1930
Dynamics of Dengue Virus (DENV)-Specific B Cells in the Response to DENV Serotype 1 Infections, Using Flow Cytometry With Labeled Virions.
Authors: Woda, Marcia and Friberg, Heather and Currier, Jeffrey R and Srikiatkhachorn, Anon and Macareo, Louis R and Green, Sharone and Jarman, Richard G and Rothman, Alan L and Mathew, Anuja
Journal: The Journal of infectious diseases (2016): 1001-9
Page updated on December 11, 2024

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Catalog Number1422
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Physical properties

Molecular weight

1141.99

Solvent

DMSO

Spectral properties

Excitation (nm)

624

Emission (nm)

640

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
HeLa cells were incubated with (+Tubulin) or without (control) mouse anti-tubulin followed by iFluor® 625 goat anti-mouse IgG conjugate.
HeLa cells were incubated with (+Tubulin) or without (control) mouse anti-tubulin followed by iFluor® 625 goat anti-mouse IgG conjugate.
HeLa cells were incubated with (+Tubulin) or without (control) mouse anti-tubulin followed by iFluor® 625 goat anti-mouse IgG conjugate.
Top) Spectral pattern was generated using a 4-laser spectral cytometer. Spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to create four distinct emission profiles, then, when combined, yielded the overall spectral signature. Bottom) Flow cytometry analysis of whole blood stained with PE/iFlour® 625 anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the B7-A channel.

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