ReadiCleave™ iFluor 546 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	1494.76
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.25
Correction Factor (280 nm)	0.15
Extinction coefficient (cm ^-1 M ^-1)	100000¹
Excitation (nm)	541
Emission (nm)	557
Quantum yield	0.67¹

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

1494.76

Correction Factor (260 nm)

0.25

Correction Factor (280 nm)

0.15

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

100000¹

Excitation (nm)

541

Emission (nm)

557

Quantum yield

0.67¹

Fluorescence-based methods have many advantages for biological detections in terms of sensitivity and convenience. Many biological molecules can be readily labeled with a fluorescent tag for fluorescence imaging and flow cytometry analysis. However, most of 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® 546 AML contains an azidomethyl linker that can be cleaved with TCEP to remove the iFluor® 546 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® 546 is a superior replacement to Alexa Fluor® 546. iFluor® 546 and Alexa Fluor® 546 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 546 AML-NHS ester stock solution (Solution B)

Add anhydrous DMSO into the vial of ReadiCleave™ iFluor 546 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 546 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 546 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	66.9 µL	334.502 µL	669.004 µL	3.345 mL	6.69 mL
5 mM	13.38 µL	66.9 µL	133.801 µL	669.004 µL	1.338 mL
10 mM	6.69 µL	33.45 µL	66.9 µL	334.502 µL	669.004 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

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Spectral properties

Correction Factor (260 nm)	0.25
Correction Factor (280 nm)	0.15
Extinction coefficient (cm ^-1 M ^-1)	100000¹
Excitation (nm)	541
Emission (nm)	557
Quantum yield	0.67¹

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 594 AML-NHS ester	587	603	200000¹	0.53¹	0.05	0.04

Images

Figure 1. Chemical structure for ReadiCleave™ iFluor 546 AML-NHS ester.

References

View all 40 references: Citation Explorer

Evaluation of Blood-Brain Barrier Integrity Using Vascular Permeability Markers: Evans Blue, Sodium Fluorescein, Albumin-Alexa Fluor Conjugates, and Horseradish Peroxidase.
Authors: Ahishali, Bulent and Kaya, Mehmet
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 87-103

Effects of Viscosity and Refractive Index on the Emission and Diffusion Properties of Alexa Fluor 405 Using Fluorescence Correlation and Lifetime Spectroscopies.
Authors: van Zanten, Camila and Melnikau, Dzmitry and Ryder, Alan G
Journal: Journal of fluorescence (2021): 835-845

Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series*.
Authors: Gebhardt, Christian and Lehmann, Martin and Reif, Maria M and Zacharias, Martin and Gemmecker, Gerd and Cordes, Thorben
Journal: Chemphyschem : a European journal of chemical physics and physical chemistry (2021)

Hot-Band Anti-Stokes Fluorescence Properties of Alexa Fluor 568.
Authors: Gajdos, Tamás and Hopp, Béla and Erdélyi, Miklós
Journal: Journal of fluorescence (2020): 437-443

Photo-isomerization of the Cyanine Dye Alexa-Fluor 647 (AF-647) in the Context of dSTORM Super-Resolution Microscopy.
Authors: Karlsson, Joshua K G and Laude, Alex and Hall, Michael J and Harriman, Anthony
Journal: Chemistry (Weinheim an der Bergstrasse, Germany) (2019): 14983-14998

Observing the Reversible Single Molecule Electrochemistry of Alexa Fluor 647 Dyes by Total Internal Reflection Fluorescence Microscopy.
Authors: Fan, Sanjun and Webb, James E A and Yang, Ying and Nieves, Daniel J and Gonçales, Vinicius R and Tran, Jason and Hilzenrat, Geva and Kahram, Mohaddeseh and Tilley, Richard D and Gaus, Katharina and Gooding, J Justin
Journal: Angewandte Chemie (International ed. in English) (2019): 14495-14498

Temporal Distribution Patterns of Alexa Fluor 647-Conjugated CeNPs in the Mouse Retina After a Single Intravitreal Injection.
Authors: Wong, Lily L and Barkam, Swetha and Seal, Sudipta and McGinnis, James F
Journal: Advances in experimental medicine and biology (2019): 125-130

Photobleaching Comparison of R-Phycoerythrin-Streptavidin and Streptavidin-Alexa Fluor 568 in a Breast Cancer Cell Line.
Authors: Ostad, Seyed Nasser and Babaei, Sepideh and Bayat, Ali Ahmad and Mahmoudian, Jafar
Journal: Monoclonal antibodies in immunodiagnosis and immunotherapy (2019): 25-29

A combined solvatochromic shift and TDDFT study probing solute-solvent interactions of blue fluorescent Alexa Fluor 350 dye: Evaluation of ground and excited state dipole moments.
Authors: Patil, Mallikarjun K and Kotresh, M G and Inamdar, Sanjeev R
Journal: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy (2019): 142-152

Comparison between photostability of Alexa Fluor 448 and Alexa Fluor 647 with conventional dyes FITC and APC by flow cytometry.
Authors: Rai, S and Bhardwaj, U and Misra, A and Singh, S and Gupta, R
Journal: International journal of laboratory hematology (2018): e52-e54