Calculator
HIS Lite™ Cy5 Bis NTA-Ni Complex
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
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
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.
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 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.
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.
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
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 66.665 µL | 333.327 µL | 666.653 µL | 3.333 mL | 6.667 mL |
| 5 mM | 13.333 µL | 66.665 µL | 133.331 µL | 666.653 µL | 1.333 mL |
| 10 mM | 6.667 µL | 33.333 µL | 66.665 µL | 333.327 µL | 666.653 µL |
Molarity calculator
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
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™ Cy3 Bis NTA-Ni Complex | 555 | 569 | 1500001 | 0.151 | 0.07 | 0.073 | - | - |
| HIS Lite™ Cy5 Tris NTA-Ni Complex | 651 | 670 | 2500001 | 0.271, 0.42 | 0.02 | 0.03 | 0.009 | 0.09 |
Citations
Authors: Duraj-Thatte, Anna M and Courchesne, Noémie-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
Authors: de Costa F, Barber CJ, Pujara PT, Reed DW, Covello PS.
Journal: Methods Mol Biol (2016): 91
Authors: Schwendener S, Perreten V.
Journal: Appl Environ Microbiol (2015): 3243
Authors: Kamkaew M, Chimnaronk S.
Journal: Protein Expr Purif (2015): 43
Authors: Iwasaki T, Tokuda Y, Kotake A, Okada H, Takeda S, Kawano T, Nakayama Y.
Journal: J Control Release (2015): 115
Authors: Panek A, Pietrow O, Filipkowski P, Synowiecki J.
Journal: Acta Biochim Pol (2013): 163
Safety data sheet