HIS Lite™ Cy3 Tris NTA-Ni Complex

Name: HIS Lite™ Cy3 Tris NTA-Ni Complex
Brand: AAT Bioquest
SKU: 12620
Price: 199 USD
Availability: InStock

Cy3-Tris NTA compound is used as a sensitive fluorescent probe for detecting polyhistidine-labeled proteins in cells, solution and solid surfaces. In combination with other color tris-NTA compounds (such as #12615 and #12617), it can be used for multicolor analysis of polyhistidine-tagged proteins. Fluorescent tris-NTA compounds provide an efficient method for site-specific and stable noncovalent fluorescence labeling of polyhistidine-tagged proteins. In contrast to the transient binding of conventional mono-NTA, the multivalent interaction of tris-NTA conjugated fluorophores form a much more stable complex with polyhistidine-tagged proteins. The high selectivity of tris-NTA compounds toward cumulated histidines enable the selective labeling of proteins in cell lysates and on the surface of live cells. Fluorescent tris-NTA conjugates can be applied for the analysis of a ternary protein complex in solution and on surfaces. The transition metal ions (e.g., Ni ion)-mediated complexation of polyhistidine-labeled proteins with fluorescent tris-NTA conjugates provides a sensitive reporter for detecting and monitoring protein-protein interactions in real time.

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

HIS Lite™ Cy3 Tris NTA-Ni Complex Stock Solution

Prepare a 5 to 10 mM stock solution by adding an appropriate amount of DMSO.

Note: Store any unused stock solution at -20 °C. Avoid repeated freeze-thaw cycles and minimize light exposure.

PREPARATION OF WORKING SOLUTION

HIS Lite™ Cy3 Tris NTA-Ni Complex Working Solution

Prepare a 1 to 10 µM HIS Lite™ Cy3 Tris NTA-Ni Complex working solution in PBS.

Note: Ensure that there is sufficient working solution to fully submerge the gel. After use, discard the working solution. Do not reuse.

SAMPLE EXPERIMENTAL PROTOCOL

The following protocol should be used only as a guideline and may require optimization to better suit your specific experimental needs.

Post-run Gel Staining Protocol

Run gels based on your standard protocol.
Place the gel in a suitable container. Fix the gel in the fixing solution for 60 minutes. Note: 40% ethanol + 10% acetic acid can be used as a fixing solution.
Wash the gel twice with the ultra-pure water.
Incubate the gel in the HIS Lite™ Cy3 Tris NTA-Ni Complex working solution for 60 minutes.

Note: Be sure to fully submerge the gel in the working solution.
Remove the working solution and wash the gel twice with PBS.
Proceed to imaging the gel immediately.

For In Vitro Complex Formation

Mix the His-tagged protein solution and the HIS Lite™ Cy3 Tris NTA-Ni Complex working solution at the appropriate concentrations.

Note: Optimization of the HIS Lite™ Cy3 Tris NTA-Ni Complex to the His-tagged protein mix must be performed for better labeling.

Note: 1 to 10 µM of HIS Lite™ Cy3 Tris NTA-Ni Complex can be used as a starting concentration.

Note: The reaction can be performed in a buffer containing 50 mM HEPES/KOH, pH 7.4, 100 mM KCl, 1 mM MgCl2, 2 mM β-mercaptoethanol, 5% glycerol, or a buffer of your choice.
Mix can be incubated for 30 minutes at room temperature or 4 ℃.

Note: Optimization of the incubation time and conditions must be performed for better labeling
Mix can then be subjected to column purification or any other downstream process.

Spectrum

Open in Advanced Spectrum Viewer

Product family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
HIS Lite™ Cy3 Bis NTA-Ni Complex	555	569	150000¹	0.15¹	0.07	0.073
HIS Lite™ OG488-Tris NTA-Ni Complex	498	526	76000	-	0.31	0.12
HIS Lite™ Cy5 Tris NTA-Ni Complex	651	670	250000¹	0.27¹, 0.4²	0.02	0.03

References

View all 50 references: Citation Explorer

Optimized immobilization of single chain variable fragment antibody onto non-toxic fluorescent nanoparticles for efficient preparation of a bioprobe.
Authors: Tateo, Seigo and Shinchi, Hiroyuki and Matsumoto, Hikaru and Nagata, Nonoka and Hashimoto, Masahito and Wakao, Masahiro and Suda, Yasuo
Journal: Colloids and surfaces. B, Biointerfaces (2023): 113192

Deep learning-assisted smartphone-based portable and visual ratiometric fluorescence device integrated intelligent gel label for agro-food freshness detection.
Authors: Lu, Zhiwei and Li, Mengjiao and Chen, Maoting and Wang, Qirui and Wu, Chun and Sun, Mengmeng and Su, Gehong and Wang, Xianxiang and Wang, Yanying and Zhou, Xinguang and Ye, Jianshan and Liu, Tao and Rao, Hanbing
Journal: Food chemistry (2023): 135640

Transition Metals Induce Quenching of Monomeric Near-Infrared Fluorescent Proteins.
Authors: Zhao, Haowen and Zastrow, Melissa L
Journal: Biochemistry (2022): 494-504

Site-Specific C-Terminal Fluorescent Labeling of Tau Protein.
Authors: Bryan, Louise and Awasthi, Saurabh and Li, Yuanjie and Nirmalraj, Peter Niraj and Balog, Sandor and Yang, Jerry and Mayer, Michael
Journal: ACS omega (2022): 47009-47014

A Simplified Protocol to Incorporate the Fluorescent Unnatural Amino Acid ANAP into Xenopus laevis Oocyte-Expressed P2X7 Receptors.
Authors: Durner, Anna and Nicke, Annette
Journal: Methods in molecular biology (Clifton, N.J.) (2022): 193-216

Page updated on November 21, 2024

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Catalog Number	12620
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Physical properties

Molecular weight	2142.71
Solvent	Water

Spectral properties

Correction Factor (260 nm)	0.07
Correction Factor (280 nm)	0.073
Extinction coefficient (cm ^-1 M ^-1)	150000¹
Excitation (nm)	555
Emission (nm)	569
Quantum yield	0.15¹