HIS Lite™ OG488-Tris NTA-Ni Complex

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. This OG488-tris-NTA compound has been reported to be an sensitive fluorescent probe for detecting polyhistidine-labeled proteins in cells, solution and solid surfaces.

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™ OG488-Tris NTA-Ni Complex Stock Solution

Prepare a 5 to 10 mM stock solution by adding an appropriate amount of ddH2O.

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

PREPARATION OF WORKING SOLUTION

HIS Lite™ OG488-Tris NTA-Ni Complex Working Solution

Prepare a 1 to 10 µM HIS Lite™ OG488-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™ OG488-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™ OG488-Tris NTA-Ni Complex working solution at the appropriate concentrations.

Note: Optimization of the HIS Lite™ OG488-Tris NTA-Ni Complex to the His-tagged protein mix must be performed for better labeling.

Note: 1 to 10 µM of HIS Lite™ OG488-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.

Calculators

Common stock solution preparation

Table 1. Volume of Water needed to reconstitute specific mass of HIS Lite™ OG488-Tris 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	54.405 µL	272.023 µL	544.046 µL	2.72 mL	5.44 mL
5 mM	10.881 µL	54.405 µL	108.809 µL	544.046 µL	1.088 mL
10 mM	5.44 µL	27.202 µL	54.405 µL	272.023 µL	544.046 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 Tris 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 25 citations: Citation Explorer

Escherichia coli tRNA 2-selenouridine synthase SelU selects its prenyl substrate to accomplish its enzymatic function
Authors: Szczupak, Patrycja and Radzikowska-Cieciura, Ewa and Kulik, Katarzyna and Madaj, Rafa{\l} and Sierant, Ma{\l}gorzata and Krakowiak, Agnieszka and Nawrot, Barbara
Journal: Bioorganic Chemistry (2022): 105739

Selective binding, magnetic separation and purification of histidine-tagged protein using biopolymer magnetic core-shell nanoparticles
Authors: Zhou, Y., Yan, D., Yuan, S., Chen, Y., Fletcher, E. E., Shi, H., Han, B.
Journal: Protein Expr Purif (2018): 5-11

Quantification of Histidine-Rich Protein 3 of Plasmodium falciparum
Authors: Palani, B.
Journal: Monoclon Antib Immunodiagn Immunother (2018): 87-90

A novel reverse micellar purification strategy for histidine tagged human interferon gamma (hIFN-gamma) protein from Pichia pastoris
Authors: Prabhu, A. A., Purkayastha, A., M and al, B., Kumar, J. P., M and al, B. B., Veeranki, V. D.
Journal: Int J Biol Macromol (2018): 2512-2524

Label-Free Direct Electrical Detection of a Histidine-Rich Protein with Sub-Femtomolar Sensitivity using an Organic Field-Effect Transistor
Authors: Minamiki, T., Sasaki, Y., Tokito, S., Minami, T.
Journal: ChemistryOpen (2017): 472-475

Page updated on November 21, 2024

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