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ReadiCleave™ AML Cy5 NHS ester

Fluorescence-based methods have many advantages for biological detections in terms of sensitivity. Many biological molecules are labeled with fluorescent tags 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. AAT Bioquest’s ReadiCleave™ linkers enable fluorescent tags conjugated to a biological target from which the added fluorescent tag can be removed when needed. This ReadiCleave™ AML Cy5 contains an azidomethyl linker that can be cleaved with TCEP to remove the Cy5 fluorophore from the target molecule. The cleavage can be carried out by adding 10 mM TCEP solution (pH 7.5), and incubating at 65 °C for 1-5 min.

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™ AML Cy5 NHS ester stock solution (Solution B)
Add anhydrous DMSO into the vial of ReadiCleave™ AML Cy5 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™ AML Cy5 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
  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 ReadiCleave™ AML Cy5 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 mg0.5 mg1 mg5 mg10 mg
1 mM83.028 µL415.138 µL830.275 µL4.151 mL8.303 mL
5 mM16.606 µL83.028 µL166.055 µL830.275 µL1.661 mL
10 mM8.303 µL41.514 µL83.028 µL415.138 µL830.275 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

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
ReadiCleave™ XFD532 AML-NHS ester534553810000.6110.240.09
ReadiCleave™ FITC AML-NHS ester4985178000010.79001, 0.9520.320.35
ReadiCleave™ XFD488 AML-NHS ester499520730000.9210.30.11
ReadiCleave™ XFD546 AML-NHS ester5615721120000.7910.210.12
ReadiCleave™ Cy5-SSL-NHS ester65167025000010.271, 0.420.020.03
ReadiCleave™ XFD647 AML-NHS ester6506712390000.3310.000.03

Citations

View all 13 citations: Citation Explorer
A rapid test strip for diagnosing glycosylated hemoglobin (HbA1c) based on fluorescent affinity immunochromatography
Authors: Ang, Chaoman and Lou, Doudou and Hu, Linling and Chen, Wei and Zhu, Yefei and Guo, Zhirui and Gu, Ning and Zhang, Yu
Journal: Analytical Sciences (2018): 18P135
Thermo-sensitive hydrogel PLGA-PEG-PLGA as a vaccine delivery system for intramuscular immunization
Authors: Wang, Xiaoyan and Zhang, Yu and Xue, Wei and Wang, Hong and Qiu, Xiaozhong and Liu, Zonghua
Journal: Journal of Biomaterials Applications (2017): 923--932
Cube-shaped theranostic paclitaxel prodrug nanocrystals with surface functionalization of SPC and MPEG-DSPE for imaging and chemotherapy
Authors: Guo, Fuqiang and Shang, Jiajia and Zhao, Hai and Lai, Kangrong and Li, Yang and Fan, Zhongxiong and Hou, Zhenqing and Su, Guanghao
Journal: Colloids and Surfaces B: Biointerfaces (2017)
Light/magnetic hyperthermia triggered drug released from multi-functional thermo-sensitive magnetoliposomes for precise cancer synergetic theranostics
Authors: Guo, Yuxin and Zhang, Yang and Ma, Jinyuan and Li, Qi and Li, Yang and Zhou, Xinyi and Zhao, Dan and Song, Hua and Chen, Qing and Zhu, Xuan
Journal: Journal of Controlled Release (2017)
Affinity-Controlled Protein Encapsulation into Sub-30 nm Telodendrimer Nanocarriers by Multivalent and Synergistic Interactions
Authors: Wang, Xu and Shi, Changying and Zhang, Li and Bodman, Alexa and Guo, D and an , undefined and Wang, Lili and Hall, Walter A and Wilkens, Stephan and Luo, Juntao
Journal: Biomaterials (2016)

References

View all 21 references: Citation Explorer
Excitation of Cy5 in self-assembled lipid bilayers using optical microresonators
Authors: Freeman LM, Li S, Dayani Y, Choi HS, Malmstadt N, Armani AM.
Journal: Appl Phys Lett (2011): 143703
Theranostic cRGD-BioShuttle Constructs Containing Temozolomide- and Cy7 For NIR-Imaging and Therapy
Authors: Wiessler M, Hennrich U, Pipkorn R, Waldeck W, Cao L, Peter J, Ehemann V, Semmler W, Lammers T, Braun K.
Journal: Theranostics (2011): 381
Rational approach to select small peptide molecular probes labeled with fluorescent cyanine dyes for in vivo optical imaging
Authors: Berezin MY, Guo K, Akers W, Livingston J, Solomon M, Lee H, Liang K, Agee A, Achilefu S.
Journal: Biochemistry (2011): 2691
In vivo detection of embryonic stem cell-derived cardiovascular progenitor cells using Cy3-labeled Gadofluorine M in murine myocardium
Authors: Adler ED, Bystrup A, Briley-Saebo KC, Mani V, Young W, Giovanonne S, Altman P, Kattman SJ, Frank JA, Weinmann HJ, Keller GM, Fayad ZA.
Journal: JACC Cardiovasc Imaging (2009): 1114
Quantitative proteomics by fluorescent labeling of cysteine residues using a set of two cyanine-based or three rhodamine-based dyes
Authors: Volke D, Hoffmann R.
Journal: Electrophoresis (2008): 4516
Page updated on December 17, 2024

Ordering information

Price
Unit size1 mg
Catalog Number7000
Quantity
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Physical properties

Molecular weight

1204.42

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.02

Correction Factor (280 nm)

0.03

Correction Factor (482 nm)

0.009

Correction Factor (565 nm)

0.09

Extinction coefficient (cm -1 M -1)

2500001

Excitation (nm)

651

Emission (nm)

670

Quantum yield

0.271, 0.42

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501

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