HIS Lite™ iFluor® 568 Tris NTA-Ni Complex

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Physical properties

Molecular weight	1936.37
Solvent	Water

Spectral properties

Correction Factor (260 nm)	0.34
Correction Factor (280 nm)	0.15
Extinction coefficient (cm ^-1 M ^-1)	100000¹
Excitation (nm)	568
Emission (nm)	587
Quantum yield	0.57¹

Storage, safety and handling

Intended use	Research Use Only (RUO)
Storage	Freeze (< -15 °C); Minimize light exposure

Overview SDS Protocol

Molecular weight

1936.37

Correction Factor (260 nm)

0.34

Correction Factor (280 nm)

0.15

Extinction coefficient (cm ^-1 M ^-1)

100000¹

Excitation (nm)

568

Emission (nm)

587

Quantum yield

0.57¹

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 iFluor® 568 Tris NTA compound is used as an sensitive fluorescent probe for detecting polyhistidine-labeled proteins in cells, solution and solid surfaces. In combination with OG488-tris-NTA compound it can be used for multicolor analysis of polyhistidine-tagged proteins.

Platform

Gel Imager

Excitation	Green laser
Emission	602/50 nm

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™ iFluor® 568 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™ iFluor® 568 Tris NTA-Ni Complex Working Solution

Prepare a 1 to 10 µM HIS Lite™ iFluor® 568 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™ iFluor® 568 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™ iFluor® 568 Tris NTA-Ni Complex working solution at the appropriate concentrations.

Note: Optimization of the HIS Lite™ iFluor® 568 Tris NTA-Ni Complex to the His-tagged protein mix must be performed for better labeling.

Note: 1 to 10 µM of HIS Lite™ iFluor® 568 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™ iFluor® 568 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	51.643 µL	258.215 µL	516.43 µL	2.582 mL	5.164 mL
5 mM	10.329 µL	51.643 µL	103.286 µL	516.43 µL	1.033 mL
10 mM	5.164 µL	25.822 µL	51.643 µL	258.215 µL	516.43 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

Spectral properties

Correction Factor (260 nm)	0.34
Correction Factor (280 nm)	0.15
Extinction coefficient (cm ^-1 M ^-1)	100000¹
Excitation (nm)	568
Emission (nm)	587
Quantum yield	0.57¹

Product Family

Name	Excitation (nm)	Emission (nm)
HIS Lite™ iFluor™ 647 Tris NTA-Ni Complex	648	668

Images

Figure 1. Chemical structure for HIS Lite™ iFluor® 568 Tris NTA-Ni Complex.

Citations

View all 24 citations: Citation Explorer

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

Controlling Protein Surface Orientation by Strategic Placement of Oligo-Histidine Tags
Authors: Wasserberg, D., Cabanas-Danes, J., Prangsma, J., O'Mahony, S., Cazade, P. A., Tromp, E., Blum, C., Thompson, D., Huskens, J., Subramaniam, V., Jonkheijm, P.
Journal: ACS Nano (2017): 9068-9083

Nonfouling NTA-PEG-Based TEM Grid Coatings for Selective Capture of Histidine-Tagged Protein Targets from Cell Lysates
Authors: Benjamin, C. J., Wright, K. J., Hyun, S. H., Krynski, K., Yu, G., Bajaj, R., Guo, F., Stauffacher, C. V., Jiang, W., Thompson, D. H.
Journal: Langmuir (2016): 551-9

Ni(II)NTA AuNPs as a low-resource malarial diagnostic platform for the rapid colorimetric detection of Plasmodium falciparum Histidine-Rich Protein-2
Authors: Gulka, C. P., Swartz, J. D., Wright, D. W.
Journal: Talanta (2015): 94-101

Coffee rings as low-resource diagnostics: detection of the malaria biomarker Plasmodium falciparum histidine-rich protein-II using a surface-coupled ring of Ni(II)NTA gold-plated polystyrene particles
Authors: Gulka, C. P., Swartz, J. D., Trantum, J. R., Davis, K. M., Peak, C. M., Denton, A. J., Haselton, F. R., Wright, D. W.
Journal: ACS Appl Mater Interfaces (2014): 6257-63

High-affinity gold nanoparticle pin to label and localize histidine-tagged protein in macromolecular assemblies
Authors: Anthony, K. C., You, C., Piehler, J., Pomeranz Krummel, D. A.
Journal: Structure (2014): 628-35

Comparative assessment of different histidine-tags for immobilization of protein onto surface plasmon resonance sensorchips
Authors: Fischer, M., Leech, A. P., Hubbard, R. E.
Journal: Anal Chem (2011): 1800-7