HIS Lite™ Cy5 Tris NTA-Ni Complex

Cy5-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™ Cy5 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™ Cy5 Tris NTA-Ni Complex Working Solution

Prepare a 1 to 10 µM HIS Lite™ Cy5 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™ Cy5 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™ Cy5 Tris NTA-Ni Complex working solution at the appropriate concentrations.

Note: Optimization of the HIS Lite™ Cy5 Tris NTA-Ni Complex to the His-tagged protein mix must be performed for better labeling.

Note: 1 to 10 µM of HIS Lite™ Cy5 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™ Cy5 Bis NTA-Ni Complex	651	670	250000¹	0.27¹, 0.4²	0.02	0.03
HIS Lite™ OG488-Tris NTA-Ni Complex	498	526	76000	-	0.31	0.12
HIS Lite™ Cy3 Tris NTA-Ni Complex	555	569	150000¹	0.15¹	0.07	0.073

References

View all 9 references: Citation Explorer

Expression and purification of recombinant arginine decarboxylase (speA) from Escherichia coli.
Authors: Song, Jiaping and Zhou, Chuanwen and Liu, Rui and Wu, Xudong and Wu, Di and Hu, Xiaojian and Ding, Yu
Journal: Molecular biology reports (2010): 1823-9

[Isolation, prokaryotic expression and activity analysis of thymidylate kinase (tmk) gene from Phytoplasma of wheat blue dwarf].
Authors: Li, Bei and Ji, Lingling and Wu, Yunfeng and Hao, Xing'an
Journal: Wei sheng wu xue bao = Acta microbiologica Sinica (2008): 739-44

Bacterial elongation factors EF-Tu, their mutants, chimeric forms, and domains: isolation and purification.
Authors: Jonák, J
Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (2007): 141-53

[Displaying polyhistidine peptide on the cell surface of Bacillus thuringiensis by S-layer protein].
Authors: Wang, Li and Sun, Ming and Yu, Zi Niu
Journal: Shi yan sheng wu xue bao (2003): 476-81

Lack of antitumor activity of recombinant endostatin in a human neuroblastoma xenograft model.
Authors: Jouanneau, E and Alberti, L and Nejjari, M and Treilleux, I and Vilgrain, I and Duc, A and Combaret, V and Favrot, M and Leboulch, P and Bachelot, T
Journal: Journal of neuro-oncology (2001): 11-8

Page updated on December 17, 2024

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Catalog Number	12619
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Physical properties

Molecular weight	2168.75
Solvent	Water

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