HIS Lite™ Cy3 Bis NTA-Ni Complex

Polyhistidine is one of the most popular affinity tags incorporated into recombinant proteins. It can be inserted either at the N- or C-terminus, and expressed in a variety of hosts. Due to its small size, the polyhistidine tag serves as an elegant tool for both protein purification and detection. HIS Lite™ Cy3 Bis NTA-Ni and Cy5 Bis NTA-Ni Complexes provide specific and highly sensitive detection of His-tagged fusion proteins. The Ni-NTA complexes were first reported by Kapanidis et Al. to be specific for polyhistidine tags with minimal crossreactivity. Cy3 and Cy5 dyes demonstrate strong fluorescent signals at commonly available wavelengths and with little quenching. The Cy3 Bis NTA-Ni and Cy5 Bis NTA-Ni Complexes can be directly applied either to an SDS-PAGE gel or Western blot membrane for fluorescence imaging. Detection with the Cy3 Bis NTA-Ni and Cy5 Bis NTA-Ni Complexes requires less incubation time than for protein-antibody binding. No secondary reaction is required since the Ni-NTA complexes are directly conjugated to the fluorophores.

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 Bis NTA-Ni Complex Stock Solution

Prepare a 5 to 10 mM stock solution by adding an appropriate amount of H2O2.

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 Bis NTA-Ni Complex Working Solution

Prepare a 1 to 10 µM HIS Lite™ Cy3 Bis 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 Bis 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 Bis NTA-Ni Complex working solution at the appropriate concentrations.

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

Note: 1 to 10 µM of HIS Lite™ Cy3 Bis 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.

Calculators

Common stock solution preparation

Table 1. Volume of Water needed to reconstitute specific mass of HIS Lite™ Cy3 Bis NTA-Ni Complex 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	72.22 µL	361.099 µL	722.199 µL	3.611 mL	7.222 mL
5 mM	14.444 µL	72.22 µL	144.44 µL	722.199 µL	1.444 mL
10 mM	7.222 µL	36.11 µL	72.22 µL	361.099 µL	722.199 µL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

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)	Correction Factor (482 nm)	Correction Factor (565 nm)
HIS Lite™ Cy5 Bis NTA-Ni Complex	651	670	250000¹	0.27¹, 0.4²	0.02	0.03	0.009	0.09
HIS Lite™ Cy3 Tris NTA-Ni Complex	555	569	150000¹	0.15¹	0.07	0.073	-	-

Citations

View all 6 citations: Citation Explorer

Nanoparticle-Conjugated Toll-Like Receptor 9 Agonists Improve the Potency, Durability, and Breadth of COVID-19 Vaccines
Authors: Ou, Ben S and Baillet, Julie and Picece, Vittoria CTM and Gale, Emily C and Powell, Abigail E and Saouaf, Olivia M and Yan, Jerry and Nejatfard, Anahita and Lopez Hernandez, Hector and Appel, Eric A
Journal: ACS nano (2024)

Mutational and Environmental Effects on the Dynamic Conformational Distributions of Lys48-Linked Ubiquitin Chains
Authors: Hiranyakorn, Methanee and Yagi-Utsumi, Maho and Yanaka, Saeko and Ohtsuka, Naoya and Momiyama, Norie and Satoh, Tadashi and Kato, Koichi
Journal: International Journal of Molecular Sciences (2023): 6075

Nanoparticle-Conjugated TLR9 Agonists Improve the Potency, Durability, and Breadth of COVID-19 Vaccines
Authors: Ou, Ben S and Picece, Vittoria CTM and Baillet, Julie and Gale, Emily C and Powell, Abigail E and Saouaf, Olivia M and Yan, Jerry and Hernandez, Hector Lopez and Appel, Eric
Journal: bioRxiv (2023)

Arabidopsis cryptochrome 2 forms photobodies with TCP22 under blue light and regulates the circadian clock
Authors: Mo, Weiliang and Zhang, Junchuan and Zhang, Li and Yang, Zhenming and Yang, Liang and Yao, Nan and Xiao, Yong and Li, Tianhong and Li, Yaxing and Zhang, Guangmei and others,
Journal: Nature communications (2022): 1--15

Prolonged Codelivery of hemagglutinin and a TLR7/8 agonist in a supramolecular polymer--nanoparticle hydrogel enhances potency and breadth of influenza vaccination
Authors: Roth, Gillie A and Saouaf, Olivia M and Smith, Anton AA and Gale, Emily C and Hernandez, Marcela Alcantara and Idoyaga, Juliana and Appel, Eric A
Journal: ACS biomaterials science \& engineering (2021): 1889--1899

References

View all 14 references: Citation Explorer

Purification of a Recombinant Polyhistidine-Tagged Glucosyltransferase Using Immobilized Metal-Affinity Chromatography (IMAC)
Authors: de Costa F, Barber CJ, Pujara PT, Reed DW, Covello PS.
Journal: Methods Mol Biol (2016): 91

New shuttle vector-based expression system to generate polyhistidine-tagged fusion proteins in Staphylococcus aureus and Escherichia coli
Authors: Schwendener S, Perreten V.
Journal: Appl Environ Microbiol (2015): 3243

Cellular uptake and in vivo distribution of polyhistidine peptides
Authors: Iwasaki T, Tokuda Y, Kotake A, Okada H, Takeda S, Kawano T, Nakayama Y.
Journal: J Control Release (2015): 115

Characterization of soluble RNA-dependent RNA polymerase from dengue virus serotype 2: The polyhistidine tag compromises the polymerase activity
Authors: Kamkaew M, Chimnaronk S.
Journal: Protein Expr Purif (2015): 43

Effects of the polyhistidine tag on kinetics and other properties of trehalose synthase from Deinococcus geothermalis
Authors: Panek A, Pietrow O, Filipkowski P, Synowiecki J.
Journal: Acta Biochim Pol (2013): 163

Page updated on November 23, 2024

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Catalog Number	12610
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Physical properties

Molecular weight	1384.66
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

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	12352200