Biotin azide (29 (link)) or Cy5 azide (Lumiprobe) was added to a solution of AA3-linked 17-mer to 0.5 mM followed by the addition of a freshly prepared solution of CuBr/TBTA (1:3 in DMSO/t-BuOH 3:1, 0.5 mM, Sigma-Aldrich). The mixture was shaken at 45°C for 1 Hr. The DNAs were electrophoresed and analyzed as described above.
Cy5 azide
Manufactured by Lumiprobe
Cy5-azide is a fluorescent dye used in various biomedical applications. It has an absorption maximum at 649 nm and an emission maximum at 670 nm, making it suitable for detection with red fluorescent imaging systems. Cy5-azide can be utilized for labeling biomolecules such as proteins, nucleic acids, and other targets.
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Lab products found in correlation
Sodium ascorbate, by Merck Group (4 mentions)
Copper 2 sulfate, by Merck Group (4 mentions)
Triton x 100, by Merck Group (2 mentions)
Vinblastine, by Merck Group (1 mentions)
Cuso4, by Merck Group (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
γ interferon, by Roche (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
High capacity cdna rt kit, by Thermo Fisher Scientific (1 mentions)
Goat anti chicken 488, by Thermo Fisher Scientific (1 mentions)
T511293, by Merck Group (1 mentions)
Chicken anti gfp, by Abcam (1 mentions)
Bio dot microfiltration apparatus, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
F ara edu, by Merck Group (1 mentions)
11 protocols using cy5 azide
1
Biotin/Cy5 Azide Conjugation to 17-mer DNA
2
Tubulin Photoaffinity Profiling
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Porcine tubulin (Cytoskeleton; 10 μM; T238P-A) was incubated in the absence or presence of colchicine (TCI; C0380), vinblastine (Sigma; V1377), and Taxol (TCI; P1632). A photoaffinity-based target ID probe of 1 (5 μM) was added and incubated for 75 min at room temperature. UV light (365 nm) was irradiated for 5 min to generate a covalent bond between the probe and tubulins. A click reaction between the acetylene group of target ID probe 1 and Cy5-azide was conducted at room temperature for 1.5 h. For the click reaction, 5% t-BuOH (TCI; B0706), 1 mM CuSO4 (Sigma; #209198), 100 μM TBTA {tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, Sigma; #678937}, 2 mM TCEP {tris(2-carboxyethyl) phosphine hydrochloride, Alfa; J60316}, and 40 μM Cy5-azide (Lumiprobe; #33030) were added. Tubulin protein was analyzed by SDS‒PAGE, and Cy5-labeled tubulin was measured by a fluorescent gel scanner (Azure Biosystems; Sapphire). The whole loading level of tubulins was visualized by silver staining and imaged by ChemiDoc (Bio-Rad).
3
Copper-Catalyzed Cy5 Azide Conjugation
4
Fmoc-Based Peptide Synthesis and Cell Culture Methods
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9-Fluorenylmethoxycarbonyl (Fmoc)-protected L-α-amino acids and coupling reagents (HBTU and PyBOP) and the Fmoc-Gly-OH-preloaded Wang resin (0.19 mmol/g) were obtained from Merck (Hohenbrunn, Germany). Fmoc-l -bis-homopropargylglycine-OH (Bpg) was purchased from Chiralix (Nijmegen, Germany). Chicken embryo extract (CEE) and horse serum for cell culture were obtained from Sera Laboratories International Ltd. (West Sussex, UK). γ-Interferon was obtained from Roche Applied Science (Penzberg, Germany). The High-Capacity cDNA RT Kit was from Applied Biosystems (Warrington, UK), Power SYBR Green PCR Master Mix from Life Technology (Paisley, UK), and Primers from Integrated DNA Technologies (Leuven, Belgium). PMO was fromGene Tools, LLC (Philomath, OR). Cy5-azide was from Lumiprobe (Hallandale Beach, FL). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO).
5
Visualizing Mitotic Cells in Live Larval Retinas
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Mitotic cells were labeled in live larvae by immersion in a solution containing 0.5 mM F-ara-EdU (Yoshimatsu et al., 2016 (link)) (Sigma T511293) in system water. The duration of treatment was timed according to the experimental paradigm. Half the solution volume was replaced every other day. For visualization of EdU labeling, fixed whole retinas were permeabilized in 0.3–0.5% TritonX-100 (Sigma T8787) in 0.1 M PBS for 30 min at room temperature, and then washed three times in PBS. Click reactions were carried out in PBS solution with 10 μM Cy5-azide (Lumiprobe A2020), 2 mM copper(II) sulfate (Sigma 45,167), and 20 mM sodium ascorbate (Sigma A7631) for 1 hr at room temperature. Samples were processed for immunohistochemistry after three PBS washes.
6
EdU labeling of Zebrafish Brain
7
Fluorescent Labeling of AP Sites
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Fluorescent labelling of the uracil-derived AP sites was performed as previously described with minor modifications [45 (link)]. Briefly, genomic DNA was isolated from the ung1Δ yeast cells treated with the indicated concentrations of 5-FU or 4NQO and treated with 10 mM methoxyamine. Then, 5 μg of each DNA sample was treated with 1 unit of UDG (New England Biolabs) for 30 mins at 37°C and labeled by incubation with 5 mM AA3 for an additional hour. Following the addition of Cy5 azide (Lumiprobe) to the final concentration of 0.5 mM followed and the freshly prepared CuBr/TBTA (1:4 in DMSO/t-BuOH 3:1, 0.5 mM, Sigma), the mixture was shaken at 37°C for 2 hrs. The Cy5/AA3-labeled DNA was purified by ethanol precipitation, heated at 95°C, and transferred to a positively charged nylon membrane using the Bio-Dot microfiltration apparatus (Biorad). The membrane was scanned using the ChemiDoc MP imaging system (Biorad) with a Cy5 filter and quantified using the Image Lab software.
8
EdU Labeling and Fluorescent Imaging
9
EdU Labeling and Fluorescent Imaging
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One hour after intraperitoneal injection of 100mg/kg EdU (7180, Setareh Biotech, Eugene, OR), whole brains were harvested from animals. Tissue sections were rinsed with Tris-buffered saline (TBS) and then incubated for 20 minutes with solution consisting of 100 mM pH 8.5 Tris, 1 mM CuSO4, 10 mM Cy5-azide (B3030, Lumiprobe, Hallandale Beach, FL) and 100 mM ascorbic acid. Information on animals used is provided in Supplementary Table 9 .
10
Quantifying Proliferating Cells in Zebrafish
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