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iFluor® 840 maleimide

Fluorescent dye maleimides (such as iFluor 840 maleimide) 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 (such as iFluor 840 maleimide) 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.
Ordering information
Price ()
Catalog Number1402
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight1548.14
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.2
Correction Factor (280 nm)0.09
Extinction coefficient (cm -1 M -1)2000001
Excitation (nm)836
Emission (nm)879
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Molecular weight
1548.14
Correction Factor (260 nm)
0.2
Correction Factor (280 nm)
0.09
Extinction coefficient (cm -1 M -1)
2000001
Excitation (nm)
836
Emission (nm)
879
In vivo fluorescence imaging uses a sensitive camera to detect fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emission at the infrared (IR) region are generally preferred. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the specificity and affinity of the probes and to modulate and amplify the signal at target sites for enhanced sensitivity. Further emerging developments are aiming to achieve high-resolution, multimodality and lifetime-based in vivo fluorescence imaging. Our iFluor® 830 is designed to label proteins and other biomolecules with infrared fluorescence. Conjugates prepared with iFluor® 830 have the excitation and emission in the IR range. iFluor® 840 dye emission is well separated from commonly used far-red fluorophores such as Cy5, Cy7 or allophycocyanin (APC), facilitating multicolor analysis. This fluorophore is also useful for small animal in-vivo imaging applications or for other imaging applications that require IR detections. iFluor® 840 maleimide is thiol-reactive, can be readily used for thiol-containing biomolecules.

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.

1. iFluor™ 840 maleimide stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ 840 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.


2. 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™ 840 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. 

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of iFluor® 840 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 mM64.594 µL322.968 µL645.936 µL3.23 mL6.459 mL
5 mM12.919 µL64.594 µL129.187 µL645.936 µL1.292 mL
10 mM6.459 µL32.297 µL64.594 µL322.968 µL645.936 µL

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


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.2
Correction Factor (280 nm)0.09
Extinction coefficient (cm -1 M -1)2000001
Excitation (nm)836
Emission (nm)879

Citations


View all 8 citations: Citation Explorer
Block Face Scanning Electron Microscopy of Fluorescently Labeled Axons Without Using Near Infra-Red Branding
Authors: Maclachlan, C., Sahlender, D. A., Hayashi, S., Molnar, Z., Knott, G.
Journal: Front Neuroanat (2018): 88
Bioactive magnetic near Infra-Red fluorescent core-shell iron oxide/human serum albumin nanoparticles for controlled release of growth factors for augmentation of human mesenchymal stem cell growth and differentiation
Authors: Levy, I., Sher, I., Corem-Salkmon, E., Ziv-Polat, O., Meir, A., Treves, A. J., Nagler, A., Kalter-Leibovici, O., Margel, S., Rotenstreich, Y.
Journal: J Nanobiotechnology (2015): 34
Novel near infra-red fluorescent pH sensors based on 1-aminoperylene bisimides covalently grafted onto poly(acryloylmorpholine)
Authors: Aigner, D., Borisov, S. M., Petritsch, P., Klimant, I.
Journal: Chem Commun (Camb) (2013): 2139-41
In vitro and ex vivo evaluation of smart infra-red fluorescent caspase-3 probes for molecular imaging of cardiovascular apoptosis
Authors: Debunne, M., Portal, C., Delest, B., Brakenhielm, E., Lallem and , F., Henry, J. P., Ligeret, H., Noack, P., Massonneau, M., Romieu, A., Renard, P. Y., Thuillez, C., Richard, V.
Journal: Int J Mol Imaging (2011): 413290
Simple sensitive and simultaneous high-performance liquid chromatography method of glucoconjugated and non-glucoconjugated porphyrins and chlorins using near infra-red fluorescence detection
Authors: Canada-Canada, F., Bautista-Sanchez, A., Taverna, M., Prognon, P., Maillard, P., Grierson, D. S., Kasselouri, A.
Journal: J Chromatogr B Analyt Technol Biomed Life Sci (2005): 166-72
An infra-red light-transmitting aperture controller for use in single-cell fluorescence photometry
Authors: Mahaut-Smith, M. P.
Journal: J Microsc (1998): 60-6
Photosynthetic induction in C4 leaves : An investigation using infra-red gas analysis and chlorophyll a fluorescence
Authors: Furbank, R. T., Walker, D. A.
Journal: Planta (1985): 75-83
Fluorescent infra-red angiography of the fundus oculi using indocyanine green dye
Authors: Chopdar, A., Turk, A. M., Hill, D. W.
Journal: Trans Ophthalmol Soc U K (1978): 142-6

References


View all 19 references: Citation Explorer
Nanovesicle delivery to the liver via retinol binding protein and platelet-derived growth factor receptors: how targeting ligands affect biodistribution
Authors: Hsu, Ching-Yun and Chen, Chun-Han and Aljuffali, Ibrahim A and Dai, You-Shan and Fang, Jia-You
Journal: Nanomedicine (2017)
A target cell-specific activatable fluorescence probe for in vivo molecular imaging of cancer based on a self-quenched avidin-rhodamine conjugate
Authors: Hama Y, Urano Y, Koyama Y, Kamiya M, Bernardo M, Paik RS, Shin IS, Paik CH, Choyke PL, Kobayashi H.
Journal: Cancer Res (2007): 2791
Fluorescence imaging in vivo: recent advances
Authors: Rao J, Dragulescu-Andrasi A, Yao H.
Journal: Curr Opin Biotechnol (2007): 17
Ex vivo fluorescence imaging of normal and malignant urothelial cells to enhance early diagnosis
Authors: Steenkeste K, Lecart S, Deniset A, Pernot P, Eschwege P, Ferlicot S, Leveque-Fort S, Bri and et R, Fontaine-Aupart MP.
Journal: Photochem Photobiol (2007): 1157
In vivo monitoring the fate of Cy5.5-Tat labeled T lymphocytes by quantitative near-infrared fluorescence imaging during acute brain inflammation in a rat model of experimental autoimmune encephalomyelitis
Authors: Berger C, Gremlich HU, Schmidt P, Cannet C, Kneuer R, Hiest and P, Rausch M, Rudin M.
Journal: J Immunol Methods (2007): 65
A protocol for imaging alternative splicing regulation in vivo using fluorescence reporters in transgenic mice
Authors: Bonano VI, Oltean S, Garcia-Blanco MA.
Journal: Nat Protoc (2007): 2166
In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy
Authors: Thiberville L, Moreno-Swirc S, Vercauteren T, Peltier E, Cave C, Bourg Heckly G.
Journal: Am J Respir Crit Care Med (2007): 22
In Vivo Fluorescence Microscopic Imaging for Dynamic Quantitative Assessment of Intestinal Mucosa Permeability in Mice
Authors: Szabo A, Vollmar B, Boros M, Menger MD.
Journal: J Surg Res. (2007)
In vivo spectral fluorescence imaging of submillimeter peritoneal cancer implants using a lectin-targeted optical agent
Authors: Hama Y, Urano Y, Koyama Y, Kamiya M, Bernardo M, Paik RS, Krishna MC, Choyke PL, Kobayashi H.
Journal: Neoplasia (2006): 607
In vivo imaging of green fluorescent protein-expressing cells in transgenic animals using fibred confocal fluorescence microscopy
Authors: Al-Gubory KH, Houdebine LM.
Journal: Eur J Cell Biol (2006): 837