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HIS Lite™ Cy5 Bis NTA-Ni Complex

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Physical properties
Molecular weight1500.03
SolventWater
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 yield0.271, 0.42
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Molecular weight
1500.03
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
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.

Platform


Gel Imager

ExcitationRed laser
Emission700/50 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™ Cy5 Bis NTA-Ni Complex Stock Solution
  1. 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 Bis NTA-Ni Complex Working Solution
  1. Prepare a 1 to 10 µM HIS Lite™ Cy5 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
  1. Run gels based on your standard protocol.

  2. 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.

  3. Wash the gel twice with the ultra-pure water.

  4. Incubate the gel in the HIS Lite™ Cy5 Bis NTA-Ni Complex working solution for 60 minutes.

    Note: Be sure to fully submerge the gel in the working solution.

  5. Remove the working solution and wash the gel twice with PBS.

  6. Proceed to imaging the gel immediately.

For In Vitro Complex Formation
  1. Mix the His-tagged protein solution and the HIS Lite™ Cy5 Bis NTA-Ni Complex working solution at the appropriate concentrations.

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

    Note: 1 to 10 µM of HIS Lite™ Cy5 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.

  2. 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

  3. 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™ Cy5 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 mg0.5 mg1 mg5 mg10 mg
1 mM66.665 µL333.327 µL666.653 µL3.333 mL6.667 mL
5 mM13.333 µL66.665 µL133.331 µL666.653 µL1.333 mL
10 mM6.667 µL33.333 µL66.665 µL333.327 µL666.653 µL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

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 yield0.271, 0.42

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)Correction Factor (482 nm)Correction Factor (565 nm)
HIS Lite™ Cy3 Bis NTA-Ni Complex55556915000010.1510.070.073--
HIS Lite™ Cy5 Tris NTA-Ni Complex65167025000010.271, 0.420.020.030.0090.09

Images


Citations


View all 1 citations: Citation Explorer
Genetically Programmable Self-Regenerating Bacterial Hydrogels
Authors: Duraj-Thatte, Anna M and Courchesne, No&eacute;mie-Manuelle Dorval and Praveschotinunt, Pichet and Rutledge, Jarod and Lee, Yuhan and Karp, Jeffrey M and Joshi, Neel S
Journal: Advanced Materials (2019): 1901826

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
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
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
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
Electrodeposition of polymer nanodots with controlled density and their reversible functionalization by polyhistidine-tag proteins
Authors: Bazin D, Chevalier S, Saadaoui H, Santarelli X, Larpent C, Feracci H, Faure C.
Journal: Langmuir (2012): 13968
Surface-attached polyhistidine-tag proteins characterized by FTIR difference spectroscopy
Authors: Pinkerneil P, Guldenhaupt J, Gerwert K, Kotting C.
Journal: Chemphyschem (2012): 2649
A pair of ligation-independent Escherichia coli expression vectors for rapid addition of a polyhistidine affinity tag to the N- or C-termini of recombinant proteins
Authors: Dan H, Balach and ran A, Lin M.
Journal: J Biomol Tech (2009): 241
A facile method for reversibly linking a recombinant protein to DNA
Authors: Goodman RP, Erben CM, Malo J, Ho WM, McKee ML, Kapanidis AN, Turberfield AJ.
Journal: Chembiochem (2009): 1551
RNA aptamer binding to polyhistidine-tag
Authors: Tsuji S, Tanaka T, Hirabayashi N, Kato S, Akitomi J, Egashira H, Waga I, Ohtsu T.
Journal: Biochem Biophys Res Commun (2009): 227