iFluor® 488 maleimide

Ordering information

Price
Catalog Number
Unit Size
Quantity

Add to cart

Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

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-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

Alternative formats

Overview SDS Protocol

Molecular weight

881.76

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

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.

1. iFluor™ 488 maleimide stock solution (Solution B)

Add anhydrous DMSO into the vial of iFluor™ 488 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™ 488 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.

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 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	113.41 µL	567.048 µL	1.134 mL	5.67 mL	11.341 mL
5 mM	22.682 µL	113.41 µL	226.819 µL	1.134 mL	2.268 mL
10 mM	11.341 µL	56.705 µL	113.41 µL	567.048 µL	1.134 mL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

Open in Advanced Spectrum Viewer

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

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® 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® 488 tyramide	491	516	75000¹	0.9¹	0.21	0.11
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	588	604	180000¹	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® 488 Styramide Superior Replacement for Alexa Fluor 488 tyramide and Opal 520	491	516	75000¹	0.9¹	0.21	0.11
iFluor® 460 maleimide	468	493	80000¹	~0.8¹	0.98	0.46
iFluor® 488 TCO	491	516	75000¹	0.9¹	0.21	0.11
iFluor® 488 Tetrazine	491	516	75000¹	0.9¹	0.21	0.11
iFluor® 665 maleimide	667	692	110,000¹	0.22¹	0.12	0.09
iFluor®488-dUTP 1 mM in Tris Buffer (pH 7.5)	491	516	75000¹	0.9¹	0.21	0.11
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
ATTO 488 maleimide	499	520	90000	0.80	0.25	0.10
Show More (25)

Images

Figure 1. 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.

Figure 2. Fluorescence In Situ Hybridization of Fluorescein and iFluor® 488 labelled Telomere probes in metaphase HeLa cells.

Citations

View all 12 citations: Citation Explorer

Kukoamine A attenuates lipopolysaccharide-induced apoptosis, extracellular matrix degradation, and inflammation in nucleus pulposus cells by activating the P13K/Akt pathway
Authors: Wang, Dan and Qu, Hao and Kang, Hui and Xu, Feng and Huang, Wei and Cai, Xianhua
Journal: Bioengineered (2022): 8772--8784

Independent phenotypic plasticity axes define distinct obesity sub-types
Authors: Yang, Chih-Hsiang and Fagnocchi, Luca and Apostle, Stefanos and Wegert, Vanessa and Casan{\'\i}-Gald{\'o}n, Salvador and Landgraf, Kathrin and Panzeri, Ilaria and Dror, Erez and Heyne, Steffen and W{\"o}rpel, Till and others,
Journal: Nature metabolism (2022): 1150--1165

ZBTB7A promotes virus-host homeostasis during human coronavirus 229E infection
Authors: Zhu, Xinyu and Trimarco, Joseph D and Williams, Courtney A and Barrera, Alejandro and Reddy, Timothy E and Heaton, Nicholas S
Journal: Cell Reports (2022): 111540

Osteoclasts directly influence castration-resistant prostate cancer cells
Authors: Huang, Junchi and Freyhult, Eva and Buckland, Robert and Josefsson, Andreas and Damber, Jan-Erik and Wel{\'e}n, Karin
Journal: Clinical \& experimental metastasis (2022): 801--814

A Novel CDK4/6 and PARP Dual Inhibitor ZC-22 Effectively Suppresses Tumor Growth and Improves the Response to Cisplatin Treatment in Breast and Ovarian Cancer
Authors: Tian, Chenchen and Wei, Yufan and Li, Jianjun and Huang, Zhi and Wang, Qiong and Lin, Yingxue and Lv, Xingping and Chen, Yanan and Fan, Yan and Sun, Peiqing and others,
Journal: International journal of molecular sciences (2022): 2892

Endogenous glutamine is rate-limiting for anti-Cd3 and anti-Cd28 induced Cd4+ T-cell proliferation and glycolytic activity under hypoxia and normoxia
Authors: Wik, Jonas A and Chowdhury, Azazul and Kolan, Shrikant and Bastani, Nasser E and Li, Gaoyang and Alam, Kazi and Grimolizzi, Franco and Sk{\aa}lhegg, Bj{\o}rn S
Journal: Biochemical Journal (2022): 1221--1235

Regulation of circADAMTS6-miR-324-5p-PIK3R3 ceRNA pathway may be a novel mechanism of IL-1$\beta$-induced osteoarthritic chondrocytes
Authors: Shen, Lanjuan and Ji, Cheng and Lin, Jian and Yang, Hongping
Journal: Journal of Bone and Mineral Metabolism (2022): 389--401

Silencing of Circ\_0135889 Restrains Proliferation and Tumorigenicity of Human Neuroblastoma Cells
Authors: Yang, Jun and Liu, Bao and Xu, Zhenli and Feng, Mei
Journal: Journal of Surgical Research (2022): 135--147

CD95/Fas protects triple negative breast cancer from anti-tumor activity of NK cells
Authors: Qadir, Abdul S and Gu{\'e}gan, Jean Philippe and Ginestier, Christophe and Chaibi, Assia and Bessede, Alban and Charafe-Jauffret, Emmanuelle and Macario, Manon and Lavou{\'e}, Vincent and de la Motte Rouge, Thibault and Law, Calvin and others,
Journal: Iscience (2021): 103348

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

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

Alexa Fluor 546-ArIB[V11L;V16A] is a potent ligand for selectively labeling alpha 7 nicotinic acetylcholine receptors
Authors: Hone AJ, Whiteaker P, Mohn JL, Jacob MH, McIntosh JM.
Journal: J Neurochem (2010): 994

Asymmetric trimethine 3H-indocyanine dyes: efficient synthesis and protein labeling
Authors: Song F, Wang L, Qiao X, Wang B, Sun S, Fan J, Zhang L, Peng X.
Journal: Org Biomol Chem (2010): 4249

Neuroanatomical characteristics of acupoint "Chengshan" (BL 57) in the rat: a cholera toxin subunit B conjugated with Alexa Fluor 488 method study
Authors: Zhu XL, Bai WZ, Wu FD, Jiang J, Jing XH.
Journal: Zhen Ci Yan Jiu (2010): 433

Photoactivatable and photoconvertible fluorescent probes for protein labeling
Authors: Maurel D, Banala S, Laroche T, Johnsson K.
Journal: ACS Chem Biol (2010): 507

Novel Alexa Fluor-488 labeled antagonist of the A(2A) adenosine receptor: Application to a fluorescence polarization-based receptor binding assay
Authors: Kecskes M, Kumar TS, Yoo L, Gao ZG, Jacobson KA.
Journal: Biochem Pharmacol (2010): 506