iFluor® 460 maleimide

Name: iFluor® 460 maleimide
Brand: AAT Bioquest
SKU: 1058
Price: 216 USD
Availability: InStock

AAT Bioquest's iFluor® dyes are optimized for labeling proteins, particularly antibodies. These dyes are bright, photostable, and have minimal quenching on proteins. Although the 460 nm blue diode laser is being installed in numerous new fluorescence instruments, few dyes can be well excited at 460 nm. iFluor® 460 is optimized to be well excited by the blue diode laser at 460 nm, enabling new biological applications for the new fluorescence instruments equipped with the 460 nm blue diode laser. iFluor® 460 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.

1. iFluor™ 460 maleimide stock solution (Solution B)

Add anhydrous DMSO into the vial of iFluor™ 460 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:

Prepare a fresh solution of 1 M DTT (15.4 mg/100 µL) in distilled water.
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).
Pass the reduced IgG over a filtration column pre-equilibrated with "Exchange Buffer". Collect 0.25 mL fractions off the column.
Determine the protein concentrations and pool the fractions with the majority of the IgG. This can be done either spectrophotometrically or colorimetrically.
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™ 460 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

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.
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.

Prepare Sephadex G-25 column according to the manufacture instruction.
Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
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.

Spectrum

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Product family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
iFluor® 350 maleimide	345	450	20000¹	0.95¹	0.83	0.23
iFluor® 488 maleimide	491	516	75000¹	0.9¹	0.21	0.11
iFluor® 555 maleimide	557	570	100000¹	0.64¹	0.23	0.14
iFluor® 647 maleimide	656	670	250000¹	0.25¹	0.03	0.03
iFluor® 680 maleimide	684	701	220000¹	0.23¹	0.097	0.094
iFluor® 700 maleimide	690	713	220000¹	0.23¹	0.09	0.04
iFluor® 750 maleimide	757	779	275000¹	0.12¹	0.044	0.039
iFluor® 790 maleimide	787	812	250000¹	0.13¹	0.1	0.09
iFluor® 800 maleimide	801	820	250000¹	0.11¹	0.03	0.08
iFluor® 810 maleimide	811	822	250000¹	0.05¹	0.09	0.15
iFluor® 820 maleimide	822	850	250000¹		0.11	0.16
iFluor® 860 maleimide	853	878	250000¹		0.1	0.14
iFluor® 532 maleimide	537	560	90000¹	0.68¹	0.26	0.16
iFluor® 594 maleimide	587	603	200000¹	0.53¹	0.05	0.04
iFluor® 405 maleimide	403	427	37000¹	0.91¹	0.48	0.77
iFluor® 430 maleimide	433	498	40000¹	0.78¹	0.68	0.3
iFluor® 568 maleimide	568	587	100000¹	0.57¹	0.34	0.15
iFluor® 633 maleimide	640	654	250000¹	0.29¹	0.062	0.044
iFluor® 450 maleimide	451	502	40000¹	0.82¹	0.45	0.27
iFluor® 665 maleimide	667	692	110,000¹	0.22¹	0.12	0.09
iFluor® 460 Styramide	468	493	80000¹	~0.8¹	0.98	0.46
iFluor® 546 maleimide	541	557	100000¹	0.67¹	0.25	0.15
iFluor® 840 maleimide	836	879	200000¹	-	0.2	0.09
iFluor® 770 maleimide	777	797	250000¹	0.16	0.09	0.08
iFluor® 780 maleimide	784	808	250000¹	0.16¹	0.13	0.12
iFluor® 830 maleimide	830	867	-	-	-	-
iFluor® 514 maleimide	511	527	75000¹	0.83¹	0.265	0.116
iFluor® 660 maleimide	663	678	250000¹	0.26¹	0.07	0.08
iFluor® 670 maleimide	671	682	200000¹	0.55¹	0.03	0.033
iFluor® 720 maleimide	716	740	240000¹	0.14¹	0.15	0.13
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References

View all 50 references: Citation Explorer

Toward the Clinical Development and Validation of a Thy1-Targeted Ultrasound Contrast Agent for the Early Detection of Pancreatic Ductal Adenocarcinoma.
Authors: Bam, Rakesh and Daryaei, Iman and Abou-Elkacem, Lotfi and Vilches-Moure, Jose G and Meuillet, Emmanuelle J and Lutz, Amelie and Marinelli, Edmund R and Unger, Evan C and Gambhir, Sanjiv S and Paulmurugan, Ramasamy
Journal: Investigative radiology (2020)

Tracking the physical stability of fluorescent-labeled mAbs under physiologic in vitro conditions in human serum and PBS.
Authors: Schuster, Joachim and Mahler, Hanns-Christian and Koulov, Atanas and Joerg, Susanne and Racher, Andy and Huwyler, Joerg and Detampel, Pascal and Mathaes, Roman
Journal: European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenst (2020): 193-201

Folding of single-stranded circular DNA into rigid rectangular DNA accelerates its cellular uptake.
Authors: Ohtsuki, Shozo and Shiba, Yukako and Maezawa, Tatsuoki and Hidaka, Kumi and Sugiyama, Hiroshi and Endo, Masayuki and Takahashi, Yuki and Takakura, Yoshinobu and Nishikawa, Makiya
Journal: Nanoscale (2019): 23416-23422

Combined effect of propranolol, vincristine and bevacizumab on HUVECs and BJ cells.
Authors: Bota, Mădălina and Fischer-Fodor, Eva and Bochiș, Ovidiu-Vasile and Cenariu, Mihai and Popa, Gheorghe and Blag, Cristina Lucia and Tătaru, Alexandru
Journal: Experimental and therapeutic medicine (2019): 307-315

Cell-type-specific quantification of protein synthesis in vivo.
Authors: Hidalgo San Jose, Lorena and Signer, Robert A J
Journal: Nature protocols (2019): 441-460

Page updated on September 18, 2024

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Physical properties

Molecular weight	831.88
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.98
Correction Factor (280 nm)	0.46
Extinction coefficient (cm ^-1 M ^-1)	80000¹
Excitation (nm)	468
Emission (nm)	493
Quantum yield	~0.8¹

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501