ReadiCleave™ iFluor 594 AML-NHS ester

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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	1509.77
Solvent	DMSO

Spectral properties

Absorbance (nm)	587
Correction Factor (260 nm)	0.05
Correction Factor (280 nm)	0.04
Extinction coefficient (cm ^-1 M ^-1)	200000¹
Excitation (nm)	587
Emission (nm)	603
Quantum yield	0.53¹

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

Overview SDS Protocol

Molecular weight

1509.77

Absorbance (nm)

587

Correction Factor (260 nm)

0.05

Correction Factor (280 nm)

0.04

Extinction coefficient (cm ^-1 M ^-1)

200000¹

Excitation (nm)

587

Emission (nm)

603

Quantum yield

0.53¹

Many biological molecules can be readily labeled with a fluorescent tag for fluorescence imaging and flow cytometry analysis. However, almost all the existing fluorescent tags are used to permanently labeling biological targets from which the added fluorescent tags cannot be cleaved for further downstream analysis, such as mass spectral analysis or another detection mode. AAT Bioquest’s ReadiCleave™ linkers enable fluorescent tags conjugated to a biological target from which the added fluorescent tag can be removed when needed. ReadiCleave™ iFluor® 594 AML uses an azidomethyl linker that can be cleaved with TCEP to remove the iFluor® 594 fluorophore from the target molecule. The cleavage can be carried out by adding 10-100 mM TCEP solution (pH 7.5) and incubating at 65 °C for 1-5 min. iFluor® 594 is a superior replacement to Alexa Fluor® 594. iFluor® 594 and Alexa Fluor® 594 have very similar spectral properties.

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. Protein stock solution (Solution A)

Mix 100 µL of a reaction buffer (e.g., 1 M sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) 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 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer.
Note     The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.
Note     Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results.
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.

2. ReadiCleave™ iFluor 594 AML-NHS ester stock solution (Solution B)

Add anhydrous DMSO into the vial of ReadiCleave™ iFluor 594 AML-NHS ester 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 two weeks when kept from light and moisture. Avoid freeze-thaw cycles.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with ReadiCleave™ iFluor 594 AML-NHS ester. 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 ReadiCleave™ iFluor 594 AML-NHS ester 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	66.235 µL	331.176 µL	662.353 µL	3.312 mL	6.624 mL
5 mM	13.247 µL	66.235 µL	132.471 µL	662.353 µL	1.325 mL
10 mM	6.624 µL	33.118 µL	66.235 µL	331.176 µL	662.353 µL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
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Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Absorbance (nm)	587
Correction Factor (260 nm)	0.05
Correction Factor (280 nm)	0.04
Extinction coefficient (cm ^-1 M ^-1)	200000¹
Excitation (nm)	587
Emission (nm)	603
Quantum yield	0.53¹

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
ReadiCleave™ iFluor 700 AML-NHS ester	690	713	220000¹	0.23¹	0.09	0.04
ReadiCleave™ iFluor 488 AML-NHS ester	491	516	75000¹	0.9¹	0.21	0.11
ReadiCleave™ iFluor 546 AML-NHS ester	541	557	100000¹	0.67¹	0.25	0.15

Images

Figure 1. Chemical structure for ReadiCleave™ iFluor 594 AML-NHS ester.

References

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MicroRNA-126 inhibits pathological retinal neovascularization via suppressing vascular endothelial growth factor expression in a rat model of retinopathy of prematurity.
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Journal: European journal of pharmacology (2021): 174035

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Journal: Brain structure & function (2021)

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Journal: Talanta (2021): 122039

Liquid Droplet Formation and Facile Cytosolic Translocation of IgG in the Presence of Attenuated Cationic Amphiphilic Lytic Peptides.
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Journal: Angewandte Chemie (International ed. in English) (2021)

Effect of VIRP1 Protein on Nuclear Import of Citrus Exocortis Viroid (CEVd).
Authors: Seo, Hyesu and Kim, Kyunghee and Park, Woong June
Journal: Biomolecules (2021)

Fluorescently-labeled fremanezumab is distributed to sensory and autonomic ganglia and the dura but not to the brain of rats with uncompromised blood brain barrier.
Authors: Noseda, Rodrigo and Schain, Aaron J and Melo-Carrillo, Agustin and Tien, Jason and Stratton, Jennifer and Mai, Fanny and Strassman, Andrew M and Burstein, Rami
Journal: Cephalalgia : an international journal of headache (2020): 229-240

Modeling iontophoretic drug delivery in a microfluidic device.
Authors: Moarefian, Maryam and Davalos, Rafael V and Tafti, Danesh K and Achenie, Luke E and Jones, Caroline N
Journal: Lab on a chip (2020): 3310-3321

Cell-based immunofluorescence assay for screening the neurogenesis potential of new drugs in adult hippocampal neural progenitor cells.
Authors: Zhang, Kun and Li, Bin and Li, Peifang and Yang, Xiaoli and Cui, Huixian and Liu, Xiaoyun
Journal: Acta neurobiologiae experimentalis (2019): 302-308

Super blinking and biocompatible nanoprobes based on dye doped BSA nanoparticles for super resolution imaging.
Authors: Zong, Shenfei and Pan, Fengmei and Zhang, Ruohu and Chen, Chen and Wang, Zhuyuan and Cui, Yiping
Journal: Nanotechnology (2019): 065701

[Sepsis impairs aggregation of nicotinic acetylcholine receptors on murine skeletal muscle cell membranes by inhibiting AKT/GSK3β phosphorylation].
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Journal: Nan fang yi ke da xue xue bao = Journal of Southern Medical University (2019): 1337-1343