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iFluor® 510 succinimidyl ester

iFluor® 510 succinimidyl ester is the most convenient form of activated iFluor® 510 derivatives that can be used for labeling proteins such as antibodies. It is a new fluorescent dye belonging to the iFluor® family of dyes. These dyes are known for their bright fluorescence, photostability, and compatibility with various imaging techniques and instruments. iFluor® 510 emits green fluorescence when excited with light in the blue to green range (around 488 to 514 nm). Its emission peak is typically around 520 to 530 nm. The number "510" in its name represents the approximate maximum excitation wavelength. Like other iFluor® dyes, iFluor® 510 is commonly used to develop a variety of conjugates in biological research and imaging applications. It can be conjugated to various biomolecules, such as antibodies, proteins, nucleic acids, and small molecules, for fluorescence microscopy, flow cytometry, immunohistochemistry, and other fluorescence-based assays. The iFluor® dyes are designed to offer enhanced performance compared to traditional dyes, with features like high quantum yields, excellent brightness, minimal background noise, and resistance to photobleaching. iFluor® 510, specifically, is suitable for applications where green fluorescence is desired.

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)

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.

iFluor® 510 SE stock solution (Solution B)

Add anhydrous DMSO into the vial of iFluor® 510 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® 510 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 needs to 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® 675 succinimidyl ester683700---0.066
iFluor® 620 succinimidyl ester621636---0.04
iFluor® 605 succinimidyl ester603623----
iFluor® 625 succinimidyl ester624640----
iFluor® 540 succinimidyl ester540557---0.105
iFluor® 445 succinimidyl ester446558----
iFluor® 500 succinimidyl ester501520800001-0.2060.088
Show More (36)

References

View all 50 references: Citation Explorer
A dual-mode optical fiber sensor for SERS and fluorescence detection in liquid.
Authors: Li, Minglu and Yan, Mingming and Xu, Ben and Zhao, Chunliu and Wang, Dongning and Wang, Yi and Chen, Huifang
Journal: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy (2023): 122267
Toward Plasmonic Neural Probes: SERS Detection of Neurotransmitters through Gold-Nanoislands-Decorated Tapered Optical Fibers with Sub-10 nm Gaps.
Authors: Zheng, Di and Pisano, Filippo and Collard, Liam and Balena, Antonio and Pisanello, Marco and Spagnolo, Barbara and Mach-Batlle, Rosa and Tantussi, Francesco and Carbone, Luigi and De Angelis, Francesco and Valiente, Manuel and de la Prida, Liset M and Ciracì, Cristian and De Vittorio, Massimo and Pisanello, Ferruccio
Journal: Advanced materials (Deerfield Beach, Fla.) (2023): e2200902
Label-free DNA detection using silver nanoprism decorated silicon nanoparticles: Effect of silicon nanoparticle size and doping levels.
Authors: Daoudi, Kais and Columbus, Soumya and Falcão, Bruno P and Pereira, Rui N and Peripolli, Suzana B and Ramachandran, Krithikadevi and Hadj Kacem, Hassen and Allagui, Anis and Gaidi, Mounir
Journal: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy (2023): 122262
Manifold Learning Enables Interpretable Analysis of Raman Spectra from Extracellular Vesicle and Other Mixtures.
Authors: Kazemzadeh, Mohammadrahim and Martinez-Calderon, Miguel and Otupiri, Robert and Artuyants, Anastasiia and Lowe, Moi M and Ning, Xia and Reategui, Eduardo and Schultz, Zachary D and Xu, Weiliang and Blenkiron, Cherie and Chamley, Lawrence W and Broderick, Neil G R and Hisey, Colin L
Journal: bioRxiv : the preprint server for biology (2023)
Toward SERS based localized thermometry of Polymer-Supported silver and gold nanostructures.
Authors: Prezgot, Daniel and Cruikshank, Jack and Makila-Boivin, Merrick and Birgani, Saro and Ianoul, Anatoli
Journal: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy (2022): 121514
Page updated on December 11, 2024

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Physical properties

Molecular weight

951.91

Solvent

DMSO

Spectral properties

Excitation (nm)

511

Emission (nm)

530

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® 510 goat anti-mouse IgG conjugate. Cells were imaged using a fluorescence microscope equipped with a FITC filter set.
HeLa cells were incubated with (+Tubulin) or without (control) mouse anti-tubulin followed by iFluor® 510 goat anti-mouse IgG conjugate. Cells were imaged using a fluorescence microscope equipped with a FITC filter set.
HeLa cells were incubated with (+Tubulin) or without (control) mouse anti-tubulin followed by iFluor® 510 goat anti-mouse IgG conjugate. Cells were imaged using a fluorescence microscope equipped with a FITC filter set.
HeLa cells were incubated with mouse anti-tubulin followed by iFluor® 510 goat anti-mouse IgG conjugate (green). Nuclei were stained with DAPI (blue).

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