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iFluor® 488 maleimide

Although FITC is still the most popular fluorescent labeling dye for preparing green fluorescent bioconjugates, there are certain limitations with FITC, such as severe photobleaching for microscope imaging and pH-sensitive fluorescence. Protein conjugates prepared with iFluor® 488 dyes are far superior to conjugates of fluorescein derivatives such as FITC. iFluor® 488 conjugates are significantly brighter than fluorescein conjugates and are much more photostable. Additionally, the fluorescence of iFluor® 488 is not affected by pH (4-10). This pH insensitivity is a major improvement over fluorescein, which emits its maximum fluorescence only at pH above 9. iFluor® 488 maleimide is reasonably stable and shows good reactivity and selectivity with the thiol group. This iFluor® 488 has spectral properties and reactivity similar to Alexa Fluor® 488 maleimide ( Alexa Fluor® is the trademark of Invitrogen).

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® 488 maleimide Stock Solution (Solution B)
  1. Add anhydrous DMSO into the vial of iFluor® 488 maleimide to make a 10 mM stock solution. Mix well by pipetting or vortex.

    Note: For optimal results, prepare the dye stock solution (Solution B) before starting the conjugation process. Remember to use it promptly, as extended storage of the dye stock solution may reduce its reactivity. Solution B can be stored in the freezer for up to 4 weeks, protected from light and moisture. Avoid freeze-thaw cycles.

Protein Stock Solution (Solution A)
  1. 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 a 1 mL protein labeling stock solution.

    Note: The pH of the protein labeling stock solution 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, it is recommended that the final protein concentration range between 2-10 mg/mL.

  2. Optional. If your protein does not already contain a free cysteine, it is necessary to treat it with either DTT or TCEP to generate a thiol group. This process is used to convert a disulfide bond into two free thiol groups. If DTT is used, it is important to remove any excess free DTT by dialysis or gel filtration prior to conjugating a dye maleimide to the protein. Below 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. To make an IgG solution in 20 mM DTT, add 20 µL of DTT stock per ml of IgG solution while mixing. Let the solution stand at room temperature for 30 minutes without additional mixing (to minimize the 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: For the best results, IgG solutions should be >4 mg/mL. If the antibody is less than 2 mg/mL, it should be concentrated. Include an extra 10% for losses on the buffer exchange column.

      Note: The reduction can be carried out in almost any buffer from pH 7 to 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 labeling Goat anti-mouse IgG with iFluor® 488 maleimide. Further optimization may be required for your specific 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) to the vial of the protein solution (95 µL of Solution A) with effective shaking. The protein concentration is ~0.05 mM, assuming the protein concentration is 10 mg/mL and the protein molecular weight is ~200KD.

    Note: We recommend using a 10:1 molar ratio of Solution B (dye) to Solution A (protein). If the ratio is too low 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 the Sephadex G-25 column according to the manufacturer's instructions.

  2. Load the reaction mixture (from the "Run Conjugation Reaction" section) 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 should 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.

Optional: Characterize the Desired Dye-Protein Conjugate

Determining the Degree of Substitution (DOS) is crucial in characterizing dye-labeled proteins. Lower DOS proteins tend to have weaker fluorescence, but higher DOS proteins may also have reduced fluorescence. For most antibodies, the optimal DOS is between 2 and 10, depending on the dye and protein properties. For effective labeling, the degree of substitution should be controlled to have 5-8 moles of iFluor® 488 maleimide to one mole of antibody. The following steps are used to determine the DOS of iFluor® 488 maleimide-labeled proteins:

  1. Measure absorption—To measure the absorption spectrum of a dye-protein conjugate, the sample concentration should be kept between 1 and 10 µM, depending on the dye's extinction coefficient.

  2. Read OD (absorbance) at 280 nm and dye maximum absorption (ƛmax = 516 nm for iFluor® 488 dyes). For most spectrophotometers, the sample (from the column fractions) must be diluted with de-ionized water so that the OD values range from 0.1 to 0.9. The O.D. (absorbance) at 280 nm is the maximum absorption of protein, while 516 nm is the maximum absorption of iFluor® 488 maleimide. To obtain accurate DOS, ensure the conjugate is free of the non-conjugated dye.

  3. Calculate DOS using our DOS calculator:

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® 488 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 mM113.41 µL567.048 µL1.134 mL5.67 mL11.341 mL
5 mM22.682 µL113.41 µL226.819 µL1.134 mL2.268 mL
10 mM11.341 µL56.705 µL113.41 µL567.048 µL1.134 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® 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® 488 tyramide4915167500010.910.210.11
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® 430 maleimide4334984000010.7810.680.3
iFluor® 568 maleimide56858710000010.5710.340.15
iFluor® 633 maleimide64065425000010.2910.0620.044
iFluor® 450 maleimide4515024000010.8210.450.27
iFluor® 488 Styramide *Superior Replacement for Alexa Fluor 488 tyramide and Opal 520*4915167500010.910.210.11
iFluor® 460 maleimide468493800001~0.810.980.46
iFluor® 488 TCO4915167500010.910.210.11
iFluor® 488 Tetrazine4915167500010.910.210.11
iFluor® 665 maleimide667692110,00010.2210.120.09
iFluor®488-dUTP *1 mM in TE Buffer (pH 7.5)*4915167500010.910.210.11
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
ATTO 488 maleimide499520900000.800.220.09
Show More (26)

Citations

View all 19 citations: Citation Explorer
IKIP downregulates THBS1/FAK signaling to suppress migration and invasion by glioblastoma cells
Authors: Zhu, Zhaoying and Hu, Yanjia and Ye, Feng and Teng, Haibo and You, Guoliang and Zeng, Yunhui and Tian, Meng and Xu, Jianguo and Li, Jin and Liu, Zhiyong and others,
Journal: Oncology Research (2024): 1173
LLPS of FXR proteins drives replication organelle clustering for $\beta$-coronaviral proliferation
Authors: Li, Meng and Hou, Yali and Zhou, Yuzheng and Yang, Zhenni and Zhao, Hongyu and Jian, Tao and Yu, Qianxi and Zeng, Fuxing and Liu, Xiaotian and Zhang, Zheng and others,
Journal: Journal of Cell Biology (2024)
Pharmacological inhibition of RAS overcomes FLT3 inhibitor resistance in FLT3-ITD+ AML through AP-1 and RUNX1
Authors: Coleman, Daniel JL and Keane, Peter and Chin, Paulynn S and Ames, Luke and Kellaway, Sophie and Blair, Helen and Khan, Naeem and Griffin, James and Holmes, Elizabeth and Maytum, Alexander and others,
Journal: iScience (2024)
A high-throughput DNA analysis method based on isothermal amplification on a suspension microarray for detecting mpox virus and viruses with comparable symptoms
Authors: Zhang, Liming and Liu, Jieyu and Huang, Shisi and Zeng, Wentao and Li, Li and Fan, Xihao and Lu, Zhuoxuan
Journal: Analytica Chimica Acta (2024): 342416
FAM81A is a postsynaptic protein that regulates the condensation of postsynaptic proteins via liquid--liquid phase separation
Authors: Kaizuka, Takeshi and Hirouchi, Taisei and Saneyoshi, Takeo and Shirafuji, Toshihiko and Collins, Mark O and Grant, Seth GN and Hayashi, Yasunori and Takumi, Toru
Journal: Plos Biology (2024): e3002006

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 October 4, 2024

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Physical properties

Molecular weight

881.76

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.21

Correction Factor (280 nm)

0.11

Extinction coefficient (cm -1 M -1)

750001

Excitation (nm)

491

Emission (nm)

516

Quantum yield

0.91

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
HeLa cells were stained with rabbit anti-tubulin followed by iFluor® 488 goat anti-rabbit IgG (H+L), and nuclei were stained with Nuclear Red™ DCS1 (Cat No. 17552).
HeLa cells were stained with rabbit anti-tubulin followed by iFluor® 488 goat anti-rabbit IgG (H+L), and nuclei were stained with Nuclear Red™ DCS1 (Cat No. 17552).
HeLa cells were stained with rabbit anti-tubulin followed by iFluor® 488 goat anti-rabbit IgG (H+L), and nuclei were stained with Nuclear Red™ DCS1 (Cat No. 17552).
Fluorescence In Situ Hybridization of Fluorescein and iFluor® 488 labelled Telomere probes in metaphase HeLa cells.