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AAT Bioquest

iFluor® 430 maleimide

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® 430 dyes are designed to be a superior replacement for Alexa Fluor® 430 labeling dye (Alexa Fluor® is the trademark of Invitrogen). Under the same conditions, iFluor® 430 dye conjugates are significantly brighter than the corresponding bioconjugates of Alexa Fluor 430 with much stronger absorption, making the iFluor 430 conjugates much more sensitive. iFluor® 430 maleimide is stable and shows good reactivity and selectivity with the thiol group.

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

iFluor™ 430 maleimide stock solution (Solution B)

Add anhydrous DMSO into the vial of iFluor™ 430 maleimide 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 upto 4 weeks when kept from light and moisture. Avoid freeze-thaw cycles.

Protein stock solution (Solution A)

Mix 100 µL of a reaction buffer (e.g., 100 mM MES buffer with pH ~6.0) 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 6.5 ± 0.5.

Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or other proteins will not be labeled well.

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.

Optional: if your protein does not contain a free cysteine, you must treat your protein with DTT or TCEP to generate a thiol group. DTT or TCEP are used for converting a disulfide bond to two free thiol groups. If DTT is used you must remove free DTT by dialysis or gel filtration before conjugating a dye maleimide to your protein. Following is a sample protocol for generating a free thiol group:

  1. Prepare a fresh solution of 1 M DTT (15.4 mg/100 µL) in distilled water.
  2. Make IgG solution in 20 mM DTT: add 20 µL of DTT stock per ml of IgG solution while mixing. Let stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
  3. Pass the reduced IgG over a filtration column pre-equilibrated with "Exchange Buffer". Collect 0.25 mL fractions off the column.
  4. Determine the protein concentrations and pool the fractions with the majority of the IgG. This can be done either spectrophotometrically or colorimetrically.
  5. Carry out the conjugation as soon as possible after this step (see Sample Experiment Protocol).

    Note: IgG solutions should be >4 mg/mL for the best results. The antibody should be concentrated if less than 2 mg/mL. Include an extra 10% for losses on the buffer exchange column.

    Note: The reduction can be carried out in almost any buffers from pH 7-7.5, e.g., MES, phosphate or TRIS buffers.

    Note: Steps 3 and 4 can be replaced by dialysis.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with iFluor™ 430 maleimide. 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 ~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.

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™ 430 maleimide to one mole of antibody. The following steps are used to determine the DOS of iFluor™ 430 maleimide 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 = 433 nm for iFluor™ 430 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 433 nm is the maximum absorption of iFluor™ 430 maleimide. 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® 430 maleimide 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 mM163.225 µL816.127 µL1.632 mL8.161 mL16.323 mL
5 mM32.645 µL163.225 µL326.451 µL1.632 mL3.265 mL
10 mM16.323 µL81.613 µL163.225 µL816.127 µL1.632 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 maleimide3454502000010.9510.830.23
iFluor® 488 maleimide4915167500010.910.210.11
iFluor® 555 maleimide55757010000010.6410.230.14
iFluor® 647 maleimide65667025000010.2510.030.03
iFluor® 680 maleimide68470122000010.2310.0970.094
iFluor® 700 maleimide69071322000010.2310.090.04
iFluor® 750 maleimide75777927500010.1210.0440.039
iFluor® 790 maleimide78781225000010.1310.10.09
iFluor® 800 maleimide80182025000010.1110.030.08
iFluor® 810 maleimide81182225000010.0510.090.15
iFluor® 820 maleimide82285025000010.110.16
iFluor® 860 maleimide85387825000010.10.14
iFluor® 532 maleimide5375609000010.6810.260.16
iFluor® 594 maleimide58760320000010.5310.050.04
iFluor® 405 maleimide4034273700010.9110.480.77
iFluor® 568 maleimide56858710000010.5710.340.15
iFluor® 633 maleimide64065425000010.2910.0620.044
iFluor® 450 maleimide4515024000010.8210.450.27
iFluor® 460 maleimide468493800001~0.810.980.46
iFluor® 665 maleimide667692110,00010.2210.120.09
iFluor® 430 Tyramide *Superior Replacement for Opal 480*4334984000010.7810.680.3
iFluor® 546 maleimide54155710000010.6710.250.15
iFluor® 840 maleimide8368792000001-0.20.09
iFluor® 770 maleimide77779725000010.160.090.08
iFluor® 780 maleimide78480825000010.1610.130.12
iFluor® 830 maleimide830867----
iFluor® 514 maleimide5115277500010.8310.2650.116
iFluor® 660 maleimide66367825000010.2610.070.08
iFluor® 670 maleimide67168220000010.5510.030.033
iFluor® 720 maleimide71674024000010.1410.150.13
iFluor® 560 maleimide56057112000010.5710.04820.069
Show More (22)

Citations

View all 3 citations: Citation Explorer
Deep Sequencing Analysis of the Eha-Regulated Transcriptome of Edwardsiella tarda Following Acidification
Authors: Gao, D and Liu, N and Li, Y and Zhang, Y and Liu, G and others, undefined
Journal: Metabolomics (Los Angel) (2017): 2153--0769
Suramin inhibits cullin-RING E3 ubiquitin ligases
Authors: Wu, Kenneth and Chong, Robert A and Yu, Qing and Bai, Jin and Spratt, Donald E and Ching, Kevin and Lee, Chan and Miao, Haibin and Tappin, Inger and Hurwitz, Jerard and others, undefined
Journal: Proceedings of the National Academy of Sciences (2016): E2011--E2018
Glycosaminoglycan mimicry by COAM reduces melanoma growth through chemokine induction and function
Authors: Piccard, Helene and Berghmans, Nele and Korpos, Eva and Dillen, Chris and Aelst, Ilse Van and Li, S and ra , undefined and Martens, Erik and Liekens, S and ra , undefined and Noppen, Sam and Damme, Jo Van and others, undefined
Journal: International Journal of Cancer (2012): E425--E436

References

View all 49 references: Citation Explorer
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
Visualizing dengue virus through Alexa Fluor labeling
Authors: Zhang S, Tan HC, Ooi EE.
Journal: J Vis Exp. (2011)
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
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
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
Page updated on December 6, 2024

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Catalog Number1054
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Physical properties

Molecular weight

612.65

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.68

Correction Factor (280 nm)

0.3

Extinction coefficient (cm -1 M -1)

400001

Excitation (nm)

433

Emission (nm)

498

Quantum yield

0.781

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
UNSPSC12171501
Fluorescent dye maleimides are the most popular tool for conjugating dyes to a peptide, protein, antibody, thiol-modified oligonucleotide or nucleic acid through their SH group. Maleimides react readily with the thiol group of proteins, thiol-modified oligonucleotides, and other thiol-containing molecules under neutral conditions. The resulting dye conjugates are quite stable.
Fluorescent dye maleimides are the most popular tool for conjugating dyes to a peptide, protein, antibody, thiol-modified oligonucleotide or nucleic acid through their SH group. Maleimides react readily with the thiol group of proteins, thiol-modified oligonucleotides, and other thiol-containing molecules under neutral conditions. The resulting dye conjugates are quite stable.
Fluorescent dye maleimides are the most popular tool for conjugating dyes to a peptide, protein, antibody, thiol-modified oligonucleotide or nucleic acid through their SH group. Maleimides react readily with the thiol group of proteins, thiol-modified oligonucleotides, and other thiol-containing molecules under neutral conditions. The resulting dye conjugates are quite stable.