Cy3 and Cy5 are used in proteomics experiments so that samples from two sources can be mixed and run together through the separation process. [19] [20] This eliminates variations due to differing experimental conditions that are inevitable if the samples were run separately. These variations make it extremely difficult, if not impossible, to use computers to automate the acquisition of the data after the separation is complete. Using these dyes makes the automation trivial. Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate. While patent protection for the standard Cy series of dyes has lapsed, the trademarked Cy naming remains in place. Consequently, dyes that are identical to Cy dyes, but called different names, are now sold. Perez-Gonzalez, C., Lafontaine, D.A. & Penedo, J.C. Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes. Front. Chem. 4 (2016).

Lietard, J., Ameur, D. & Somoza, M. M. Sequence-dependent quenching of fluorescein fluorescence on single-stranded and double-stranded DNA. Rsc Adv. 12, 5629–5637 (2022). The main application for cyanine dyes is in biological labeling. Nevertheless, there is a wide literature on both their synthesis and uses, and cyanines are common in some CD and DVD media. Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., Lewis, C. J. & Waggoner, A. S. Cyanine dye labeling reagents—sulfoindocyanine Succinimidyl Esters. Bioconjugate Chem. 4, 105–111 (1993). Nie, Q.; Li, C.; Wang, Y.; Hu, Y.; Pu, W.; Zhang, Q.; Cai, J.; Lin, Y.; Li, G.; Wang, C.; Li, L.; Dou, Y.; Zhang, J. Pathologically Triggered in Situ Aggregation of Nanoparticles for Inflammation-Targeting Amplification and Therapeutic Potentiation. Acta Pharmaceutica Sinica B, 2023, 13(1), 390–409. doi: 10.1016/j.apsb.2022.07.013These XGCGC motifs suggest that the identity of the final nucleobase may not be critical for high fluorescence. Indeed, in the 2nd–4th octiles of Cy3/Cy5 fluorescence (Fig. 3A,B), the identity of the second nucleobase appears to be as important as the terminal base and G nucleotides at the penultimate position occupy a large part of the high Cy3 and Cy5 signal intensity. In the second octile of Cy5 fluorescence (Fig. 3B), the 4th position still shows strong G preference, as strong as the 2nd position, which is likely a consequence of the presence of the GCG motif, a trimmed down version of the GCGC that can still enhance the fluorescence properties of Cy5, though less so for Cy3.

Holz, K., Schaudy, E., Lietard, J. & Somoza, M. M. Multi-level patterning nucleic acid photolithography. Nat. Commun. 10, 3805 (2019). Kretschy, N., Holik, A. K., Somoza, V., Stengele, K. P. & Somoza, M. M. Next-generation o-nitrobenzyl photolabile groups for light-directed chemistry and microarray synthesis. Angew. Chem. Int. Ed. 54, 8555–8559 (2015). Umezawa K, Matsui A, Nakamura Y, Citterio D, Suzuki K (2009). "Bright, color-tunable fluorescent dyes in the Vis/NIR region: establishment of new "tailor-made" multicolor fluorophores based on borondipyrromethene". Chemistry: A European Journal. 15 (5): 1096–106. doi: 10.1002/chem.200801906. PMID 19117043. The reaction tubules were placed in a dark place and incubated gently at room temperature for 60 minutes at intervals.For 10-15 minutes, gently reverse the reaction tubules several times to fully mix the two reactants and raise the bar efficiency. Armitage, Bruce A. (27 January 2005). "Cyanine Dye–DNA Interactions: Intercalation, Groove Binding, and Aggregation". DNA Binders and Related Subjects. Vol.253. pp.55–76. doi: 10.1007/b100442. ISBN 978-3-540-22835-6. {{ cite book}}: |journal= ignored ( help)

The molarity calculator equation

Cyanine dyes are used to label proteins, antibodies, peptides, nucleic acid probes, and any kind of other biomolecules to be used in a variety of fluorescence detection techniques: Flow cytometry, Microscopy (mainly Visible range, but also UV, IR), Microplate assays, Microarrays, as well as "light-up Probes," and in vivo imaging. [18] Nucleic acid labeling [ edit ] This Cyanine5 NHS ester (analog to Cy5 ® NHS ester) is a reactive dye for the labeling of amino-groups in peptides, proteins, and oligonucleotides. This dye requires a small amount of organic co-solvent (such as DMF or DMSO) to be used in labeling reactions (please see our recommended protocol for more details). This reagent is ideal for very cost-efficient labeling of soluble proteins as well as all kinds of peptides and oligonucleotides. This reagent also works well in organic solvents for small molecule labeling. For more sophisticated targets such as easily degradable proteins, when the use of DMF or DMSO is undesirable, consider using water-soluble sulfo-Cyanine 5 NHS ester which does not require any co-solvent, and features very similar fluorescent properties. Cy7 is a near-IR fluor that is invisible to the naked eye (excitation/emission maximum 750/776nm). It is used in in vivo imaging applications, as well as the Cy7.5 dye.

Holz, K., Lietard, J. & Somoza, M. M. High-power 365 nm UV LED mercury arc lamp replacement for photochemistry and chemical photolithography. ACS Sustain. Chem. Eng. 5, 828–834 (2017).Chemically, cyanines are a conjugated system between two nitrogen atoms; in each resonance structure, exactly one nitrogen atom is oxidized to an iminium. Typically, they form part of a nitrogenous heterocyclic system. [2] Harvey, B. J. & Levitus, M. Nucleobase-specific enhancement of Cy3 fluorescence. J. Fluoresc. 19, 443–448 (2009). The pH value of protein solution shall be 8.5±0.5. If the pH is lower than 8.0, 1 M sodium bicarbonate shall be used for adjustment. Cy3 fluoresces greenish yellow (~550 nm excitation, ~570nm emission), while Cy5 is fluorescent in the far-red region (~650 excitation, 670nm emission). [12] Cy3 can be detected by various fluorometers, imagers, and microscopes with standard filters for Tetramethylrhodamine (TRITC). Due to its high molar extinction coefficient, this dye is also easily detected by naked eye on electrophoresis gels, and in solution. Iqbal, A., Wang, L., Thompson, K. C., Lilley, D. M. J. & Norman, D. G. The structure of cyanine 5 terminally attached to double-stranded DNA: Implications for FRET studies. Biochemistry 47, 7857–7862 (2008).