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Cyanine5 maleimide

Manufactured by Lumiprobe
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Cyanine5 maleimide is a fluorescent dye used in various bioanalytical applications. It functions as a labeling agent, allowing the detection and visualization of biomolecules, such as proteins, antibodies, or nucleic acids, through fluorescence techniques.

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Lab products found in correlation

Hotstartaq master mix kit, by Qiagen (2 mentions) Qiaspin miniprep kit, by Qiagen (2 mentions) Megascript t7 high yield transcription kit, by Thermo Fisher Scientific (2 mentions) 5 endtag nucleic acid labeling system, by Vector Laboratories (1 mentions) Cyanine 5 azide, by Lumiprobe (1 mentions) Cyanine5 alkyne, by Lumiprobe (1 mentions) Cyanine5 nhs ester, by Lumiprobe (1 mentions) Cyanine5 amine, by Lumiprobe (1 mentions) Thiol modifier c6 s s, by Glen Research (1 mentions)

5 protocols using cyanine5 maleimide

1

Plasmid DNA Preparation and mRNA Synthesis

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Plasmid DNA preparations were carried out by using the QIAspin miniprep kit (QIAGEN). PCRs were carried out using the HotStarTaq master mix kit (QIAGEN). The aac(6′)-Ib mRNA was synthesized in vitro using the MEGAscript high-yield transcription T7 kit (Thermo Fisher Scientific) according to the recommendations of the supplier. Labeling of the 5′-end was carried out in the dark using the 5′ EndTag Nucleic Acid Labeling system (Vector Laboratories) with the fluorescent dye cyanine5 maleimide (Lumiprobe). Secondary structure of the aac(6′)-Ib mRNA was determined using mfold (49 (link)).
Jackson A., Jani S., Davies-Sala C., Soler-Bistué A.J., Zorreguieta A, & Tolmasky M.E. (2016). Assessment of configurations and chemistries of bridged nucleic acids-containing oligomers as external guide sequences: a methodology for inhibition of expression of antibiotic resistance genes. Biology Methods & Protocols, 1(1), bpw001.
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2

Cy5 Analog Compound Preparation

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Cy5 analogs were purchased from Lumiprobe (Cyanine5 NHS ester (#13020), Cyanine5 Maleimide (#13080), Cyanine5 Azide (#A3030), Cyanine5 Alkyne (#A30B0), Cyanine5 Hydrazide (#13070), Cyanine5 Amine (#130C0), Cyanine5 Carboxylic Acid (#13090). All Cy5 compounds were dissolved at 10 mM in DMSO and stored at -20°C.
Sargiacomo C., Stonehouse S., Moftakhar Z., Sotgia F, & Lisanti M.P. (2021). MitoTracker Deep Red (MTDR) Is a Metabolic Inhibitor for Targeting Mitochondria and Eradicating Cancer Stem Cells (CSCs), With Anti-Tumor and Anti-Metastatic Activity In Vivo. Frontiers in Oncology, 11, 678343.
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3

Plasmid DNA Purification and mRNA Synthesis

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Plasmid DNA preparations were carried out by using the QIAspin miniprep kit (QIAGEN). PCRs were carried out using the HotStarTaq master mix kit (QIAGEN). The aac(6ʹ)-Ib mRNA was synthesized in vitro using the MEGAscript high-yield transcription T7 kit (Thermo Fisher Scientific) according to the recommendations of the supplier. Labeling of the 5ʹ-end was carried out in the dark using the 5ʹ EndTag Nucleic Acid Labeling system (Vector Laboratories) with the fluorescent dye cyanine5 maleimide (Lumiprobe). Secondary structure of the aac(6ʹ)-Ib mRNA was determined using mfold [49 (link)].
Jackson A., Jani S., Davies-Sala C., Soler-Bistué A.J., Zorreguieta A, & Tolmasky M.E. (2016). Assessment of configurations and chemistries of bridged nucleic acids-containing oligomers as external guide sequences: a methodology for inhibition of expression of antibiotic resistance genes. Biology Methods & Protocols, 1(1), bpw001.
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4

Cas13 crRNA Design and Synthesis

Cited 2 times
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The crRNAs were designed using the Cas13design tool66 (link). The crRNA plasmids were cloned using standard restriction-ligation cloning. Forward and reverse oligos for each spacer (IDT) were annealed and inserted into a pHR backbone using T4 DNA Ligase (NEB). The backbone was modified from Addgene (#121514). Spacer sequences and predicted efficiency score for all crRNAs can be found in Supplementary Data 1. Some crRNAs were synthesized by Synthego using solid-phase phosphoramidite chemistry to contain 3’ Um modifications with additional 5’ Cy5 labeling67 (link). A protected 3’-thiol moiety was installed using Thiol-Modifier C6 S-S (Cat# 10-1936-90E, Glen Research). Following cleavage, deprotection and isolation, the free thiol was liberated using dithiothreitol and then reacted with Cyanine5 Maleimide (Cat# 43080, Lumiprobe) following the manufacturer’s guidelines. Purity and identity were confirmed via HPLC/ESI-MS. These are listed in Supplementary Data 1. They were used in the treatment tests as described below.
Zeng L., Liu Y., Nguyenla X.H., Abbott T.R., Han M., Zhu Y., Chemparathy A., Lin X., Chen X., Wang H., Rane D.A., Spatz J.M., Jain S., Rustagi A., Pinsky B., Zepeda A.E., Kadina A.P., Walker JA I.I.I., Holden K., Temperton N., Cochran J.R., Barron A.E., Connolly M.D., Blish C.A., Lewis D.B., Stanley S.A., La Russa M.F, & Qi L.S. (2022). Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro. Nature Communications, 13, 2766.
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5

Site-Specific Labeling of Histone H2B

Cited 1 time
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To allow site-specific labelling of histone H2B, a cysteine was introduced via site-directed mutagenesis (H2BK120C, as previously described55 (link)) using mutagenesis primers indicated in Table S1. H2BK120C was labelled with Cyanine5-maleimide (Lumiprobe cat #13080) as described previously55 (link). Labelled octamers were refolded as described above. Before chromatin reconstitution, the unlabelled and labelled octamers were combined at a molar ratio of 7:1 (unlabeled:labelled). Chromatin was then reconstituted as described above.
Uckelmann M., Levina V., Taveneau C., Han Ng X., Pandey V., Martinez J., Mendiratta S., Houx J., Boudes M., Venugopal H., Trépout S., Zhang Q., Flanigan S., Li M., Sierecki E., Gambin Y., Das P.P., Bell O., de Marco A, & Davidovich C. (2024). Dynamic PRC1-CBX8 stabilizes a porous structure of chromatin condensates. bioRxiv.
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