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AAT Bioquest

iFluor® 405 succinimidyl ester

AAT Bioquest's iFluor® dyes are optimized for labeling proteins, particularly 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, 555, and 633 nm). iFluor® 405 dyes have fluorescence excitation and emission maxima of ~403 nm and ~427 nm respectively. These spectral characteristics make them an excellent alternative to Alexa Fluor® 405 labeling dye (Alexa Fluor® is the trademark of Invitrogen). iFluor® 405 SE is reasonably stable and shows good reactivity and selectivity with protein amino groups.

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™ 405 SE stock solution (Solution B)

Add anhydrous DMSO into the vial of iFluor™ 405 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™ 405 SE. You might need further optimization for your particular proteins.

Note: 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 ~200K.

    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.

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 8-10 moles of iFluor™ 405 SE to one mole of antibody. The following steps are used to determine the DOS of iFluor™ 405 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 = 403 nm for iFluor™ 405 dyes)

For most spectrophotometers, the sample (from the column fractions) need 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 403 nm is the maximum absorption of iFluor™ 405 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

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of iFluor® 405 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 mM132.349 µL661.743 µL1.323 mL6.617 mL13.235 mL
5 mM26.47 µL132.349 µL264.697 µL1.323 mL2.647 mL
10 mM13.235 µL66.174 µL132.349 µL661.743 µL1.323 mL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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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® 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® 510 succinimidyl ester511530----
iFluor® 540 succinimidyl ester540557---0.105
iFluor® 445 succinimidyl ester446558----
iFluor® 500 succinimidyl ester501520----
Show More (36)

Citations

View all 13 citations: Citation Explorer
CLASP-mediated competitive binding in protein condensates directs microtubule growth
Authors: Jia, Xuanyan and Lin, Leishu and Guo, Siqi and Zhou, Lulu and Jin, Gaowei and Dong, Jiayuan and Xiao, Jinman and Xie, Xingqiao and Li, Yiming and He, Sicong and others,
Journal: Nature Communications (2024): 6509
Competitive binding-mediated mesoscale protein-protein interactions direct microtubule growth
Authors: Wei, Zhiyi and Jia, Xuanyan and Lin, Leishu and Guo, Siqi and Zhou, Lulu and Jin, Gaowei and Dong, Jiayuan and Xiao, Jinman and Xie, Xingqiao and Li, Yiming and others,
Journal: (2024)
PF-D-Trimer, a protective SARS-CoV-2 subunit vaccine: immunogenicity and application
Authors: Zhang, Zhihao and Zhou, Jinhu and Ni, Peng and Hu, Bing and Jolicoeur, Normand and Deng, Shuang and Xiao, Qian and He, Qian and Li, Gai and Xia, Yan and others,
Journal: npj Vaccines (2023): 38
Arc weakens synapses by dispersing AMPA receptors from postsynaptic density via modulating PSD phase separation
Authors: Chen, Xudong and Jia, Bowen and Araki, Yoichi and Liu, Bian and Ye, Fei and Huganir, Richard and Zhang, Mingjie
Journal: Cell Research (2022): 914--930
Arabidopsis cryptochrome 2 forms photobodies with TCP22 under blue light and regulates the circadian clock
Authors: Mo, Weiliang and Zhang, Junchuan and Zhang, Li and Yang, Zhenming and Yang, Liang and Yao, Nan and Xiao, Yong and Li, Tianhong and Li, Yaxing and Zhang, Guangmei and others,
Journal: Nature communications (2022): 1--15

References

View all 49 references: Citation Explorer
Visualizing dengue virus through Alexa Fluor labeling
Authors: Zhang S, Tan HC, Ooi EE.
Journal: J Vis Exp. (2011)
Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers
Authors: Kuwayama M, Shigemoto N, Oohara S, Tanizawa Y, Yamada H, Takeda Y, Matsuo T, Fukuda S.
Journal: J Microbiol Methods (2011): 119
Neuroanatomical basis of clinical joint application of "Jinggu" (BL 64, a source-acupoint) and "Dazhong" (KI 4, a Luo-acupoint) in the rat: a double-labeling study of cholera toxin subunit B conjugated with Alexa Fluor 488 and 594
Authors: Cui JJ, Zhu XL, Ji CF, Jing XH, Bai WZ.
Journal: Zhen Ci Yan Jiu (2011): 262
Fluorescent "Turn-on" system utilizing a quencher-conjugated peptide for specific protein labeling of living cells
Authors: Arai S, Yoon SI, Murata A, Takabayashi M, Wu X, Lu Y, Takeoka S, Ozaki M.
Journal: Biochem Biophys Res Commun (2011): 211
Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based beta-lactam probes
Authors: Sadhu KK, Mizukami S, Watanabe S, Kikuchi K.
Journal: Mol Biosyst (2011): 1766
Page updated on November 23, 2024

Ordering information

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Catalog Number
1021710217150171551
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Physical properties

Molecular weight

755.58

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.48

Correction Factor (280 nm)

0.77

Extinction coefficient (cm -1 M -1)

370001

Excitation (nm)

403

Emission (nm)

427

Quantum yield

0.911

Storage, safety and handling

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

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501
HL-60 cells were incubated with (Red, +) or without (Green, -) Anti-human HLA-ABC (W6/32 mAb), followed by iFluor® 405 goat anti-mouse IgG (H&amp;L). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the Pacific Blue channel (Ex/Em=405/445 nm).&nbsp;
HL-60 cells were incubated with (Red, +) or without (Green, -) Anti-human HLA-ABC (W6/32 mAb), followed by iFluor® 405 goat anti-mouse IgG (H&amp;L). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the Pacific Blue channel (Ex/Em=405/445 nm).&nbsp;
HL-60 cells were incubated with (Red, +) or without (Green, -) Anti-human HLA-ABC (W6/32 mAb), followed by iFluor® 405 goat anti-mouse IgG (H&amp;L). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the Pacific Blue channel (Ex/Em=405/445 nm).&nbsp;
Spectral signature of iFluor® 405 dye. Data acquired on a 4-laser Cytek Aurora and normal human peripheral blood cells stained with clone SK3 (CD4) conjugated to iFluor® 405 dye (Cat. No. 10042020) were used for analysis.