Reagents and solvents were purchased from Sigma Aldrich or Fisher Scientific and were used as received unless otherwise stated. 2-PMPA (compound 1 ) was purchased from Sigma Aldrich (St. Louis, MO). Ethylenediamine-core PAMAM-OH dendrimer generation 4 having 64 hydroxyl end-groups, Pharma grade (compound 3, D-OH ) was received as a methanolic solution from Dendritech. Before use, the methanol was evaporated and dendrimer was further purified to remove generational impurities by dialysis. The dendrimer was solubilized in water and dialyzed against water using dialysis membrane of 3kDa. The dialysis membranes were purchased from Spectrum Laboratories Inc. Azido-PEG-11-alcohol was purchased from Broad Pharm and Cy5 NHS ester was purchased from GE healthcare and used as received. Deuterated solvents for NMR spectroscopy such as (DMSO‑d6), methanol (CD3OD), water (D2O) and chloroform (CDCl3) were purchased from Sigma. D-Cy5 was synthesized using our previously published protocol 23 (link), 26 (link).
Cy5 nhs ester
Manufactured by GE Healthcare
Sourced in United Kingdom, Japan
Cy5-NHS ester is a fluorescent labeling reagent used for the covalent attachment of a Cy5 fluorophore to proteins and other biomolecules. It can be utilized in various biological and analytical applications that require fluorescent detection or imaging.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Gen pak fax column, by Waters Corporation (3 mentions)
Cm3050s cryostat, by Leica (2 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (2 mentions)
Dabco, by Merck Group (2 mentions)
Ab13970, by BD (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Ab9361, by Merck Group (2 mentions)
Mowiol 4 88, by Polysciences (2 mentions)
P 30 gel exclusion column, by Bio-Rad (2 mentions)
Dylight 488, by Jackson ImmunoResearch (2 mentions)
Ab112, by Abcam (2 mentions)
Cyanine3 (cy3), by GE Healthcare (2 mentions)
Cy3 nhs ester, by GE Healthcare (2 mentions)
Sodium ascorbate, by Merck Group (2 mentions)
5 hexynoic acid, by Merck Group (2 mentions)
2 pmpa, by Merck Group (1 mentions)
Chloroform cdcl3, by Merck Group (1 mentions)
Alexa fluor 488 nhs ester, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 405 nhs ester, by Thermo Fisher Scientific (1 mentions)
25 protocols using cy5 nhs ester
1
Dendrimer-Dye Conjugate Synthesis
2
Fluorescent Labeling of Lectins
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Lectins were labelled with Alexa Fluor 405 NHS Ester, Alexa Fluor 488 NHS Ester (Molecular Probes) or Cy5 NHS Ester (GE Healthcare) according to the manufacturer’s protocol for amine-reactive probes. Free dye was removed using Zeba Spin desalting columns with 7K MWCO (Thermo Fisher Scientific).
3
Fluorescent Labeling of Anti-Digoxigenin Fab
4
Multicolor Immunofluorescence Imaging Protocol
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Tissue was fixed in 4% PFA/PBS at room temperature for 10 minutes and equilibrated in 30% sucrose overnight at 4°C. Tissue was embedded in O.C.T. (TissueTek) and stored at −80°C. Sections were cut at 10 µm using a Leica CM3050S cryostat and incubated with primary antibodies overnight at 4°C. Primary antibodies were rabbit anti-TH (Abcam, ab112), chicken anti-GFP (Abcam, ab13970), rat anti-CD31 (BD Pharmingen, 553370), chicken anti-β-galactosidase (Abcam, ab9361), and mouse anti-smooth muscle actin (Sigma, A5228) used at 1:500. Mouse anti-smooth muscle actin antibody was directly conjugated to Cy5 NHS ester (GE Healthcare), and unbound dye was removed on a P-30 gel exclusion column (BioRad)35 . Incubation with secondary antibodies was 45 minutes at room temperature. Secondary antibodies were conjugated to either Alexa Fluor 488, Alexa Fluor 555 (Life Technologies), or DyLight 488 (Jackson ImmunoResearch). Staining with 4’,6-diamidino-2-phenylindole (DAPI; 1 ng/ml, Life Technologies) was performed after incubation with secondary antibodies for 5 minutes at room temperature. Sections were mounted in Mowiol 4–88 (Polysciences) with DABCO (25 mg/ml, Sigma-Aldrich) and visualized on a Zeiss Axiophot fluorescence microscope. Tissue samples from three or more animals were stained for representative data shown (Fig. 1g, h , Fig. 3a, b , and Extended Data Fig. 3a–c, f–h ).
5
Biotinylated Ribosome Labeling with Fluorescent Dye
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Biotinylated ribosomes reacted with a Cy5-NHS-ester (GE Healthcare Life Sciences, Little Chalfont, UK) or Atto488-NHS-ester functionalized dye (Atto-Tec GmbH, Siegen, Germany) in labelling buffer [50 mM Hepes-KOH (pH 7.5), 10 mM MgCl2, 100 mM KCl] for 20 min at 37 °C, using a 20-fold excess of dye to minimize the unlabelled fraction of ribosomes. The excess of dye was removed by pelleting the ribosomes through a 1.1 M sucrose cushion. After resuspending the pellet in Tico buffer, aggregates were removed by centrifugation and the supernatant was used for further experiments. The concentration of Cy5 and ribosomes was determined spectroscopically in a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, USA) using the absorption coefficients εcy5 = 2.5 × 105 M−1 cm−1 and εribos = 4.2 × 107 M−1 cm−1, respectively. The label ratio was calculated to be ~6 Cy5 dyes per ribosome.
6
Fluorescent Labeling of Tubulin for Microtubule Stabilization
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Bovine brain tubulin was labeled following previously described methods (Hyman et al., 1991 (link)). Using Cy5-NHS ester (GE Healthcare, PA15101) yielded 54–70% labeling. Using Alexa-568 NHS ester (Invitrogen, A20003) yielded 36–40% labeling. Labeling efficiency with biotin-PEG4-NHS (Thermo Scientific, A39259) was not calculated.
Single-cycled GMPCPP-stabilized microtubules were made as previously described (Gell et al., 2010 (link)). Briefly, 12 μM unlabeled tubulin + 1 μM Alexa-568 tubulin + 1 μM biotin tubulin was polymerized in BRB80 (80 mM Pipes, 1 mM EGTA, 1 mM MgCl2) in the presence of 1 mM GMPCPP (Jena Bioscience, NU-405L) for 1 hr at 37°C. For GMPCPP-stabilized microtubules without any labels, 14 μM unlabeled tubulin was polymerized. For GMPCPP-stabilized microtubules without biotin, 13 μM unlabeled tubulin + 1 μM Alexa-568 tubulin was polymerized.
Single-cycled GMPCPP-stabilized microtubules were made as previously described (Gell et al., 2010 (link)). Briefly, 12 μM unlabeled tubulin + 1 μM Alexa-568 tubulin + 1 μM biotin tubulin was polymerized in BRB80 (80 mM Pipes, 1 mM EGTA, 1 mM MgCl2) in the presence of 1 mM GMPCPP (Jena Bioscience, NU-405L) for 1 hr at 37°C. For GMPCPP-stabilized microtubules without any labels, 14 μM unlabeled tubulin was polymerized. For GMPCPP-stabilized microtubules without biotin, 13 μM unlabeled tubulin + 1 μM Alexa-568 tubulin was polymerized.
7
Fluorescent DNA-Protein Interaction Assay
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A DNA oligonucleotide with an internal amino-modified thymine (T∗) at the fourth base from the 5′ end (5′-CTGT∗AGAATCCCGGTGCCGAGGCCGCT-3′ Integrated DNA Technologies) was labeled with Cy5-NHS ester (GE Healthcare). The labeled oligonucleotide was purified by reverse-phase HPLC with a Poroshell 120 EC-C18 column (Agilent Technologies). The 147 bp 601 NPS was amplified from pDrive-601 NPS with the Cy5 labeled oligonucleotide and DNA oligonucleotide 5′-ACAGGATGTATATATCTGACACGTGCCTGGA-3′. The resulting Cy5 labeled 601 NPS DNA was purified by ion-exchange HPLC with a Gen-Pak Fax column (Waters).
PFV vDNA substrates were annealed DNA oligonucleotides oKEY616 5′-ATTGTCATGGAATTTTGTATATTGAGTGGCGCCCGAACAG-3′ and oKEY675 5′-CTGTTCGGGCGCCACTCAATATACAAAATTCCATGACA-3′ (Integrated DNA Technologies). When vDNA was modified with Cy5 or biotin, the moiety was at the 5′ end of oKEY675.
PFV vDNA substrates were annealed DNA oligonucleotides oKEY616 5′-ATTGTCATGGAATTTTGTATATTGAGTGGCGCCCGAACAG-3′ and oKEY675 5′-CTGTTCGGGCGCCACTCAATATACAAAATTCCATGACA-3′ (Integrated DNA Technologies). When vDNA was modified with Cy5 or biotin, the moiety was at the 5′ end of oKEY675.
8
Quantifying Peptides Bound to AAV2 Particles
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The quantification of the number of peptides bound per AAV2 particle was determined by using the absorption measurements. First, AAV2 particles were labeled with Cy5-NHS ester (GE Healthcare, Piscataway, NJ) for 2 hours. After purification by a gel filtration column, the number of attached dyes per AAV2 particle was then calculated by measuring the absorbance of purified Cy5-labeled AAV2 particles at 650 nm for Cy5-NHS ester (ε = 250,000 M−1 cm−1) and at 280 nm for AAV2 particles (ε = 6.61 × 106 M−1 cm−1).55 (link) A different initial ratio of AAV2 particles to FITC-labeled peptides was incubated for 30 minutes at 37 °C. After purification by a gel filtration column, the extent of peptides on AAV2 particles was then calculated by measuring the absorbance of the purified coupling products at 650 nm for Cy5 (ε = 250,000 M−1 cm−1) and at 494 nm for FITC (ε = 68,000 M−1 cm−1).
9
Preparation of Fluorescently Labeled Nucleosomal DNA
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Prior to polymerase chain reaction (PCR) amplifications of nucleosomal DNA, primers containing internal amino modified thymines (IDT, Supplementary Table S1) were labeled with Cy5-NHS ester (GE healthcare) and purified by reverse phase high-pressure liquid chromatography (RP-HPLC) on a C18 column (Agilent Technologies). Using a pair of Cy5-labled and unlabeled primers (Supplementary Table S1) with a Widom 601 nucleosome positioning sequence (601 NPS, Supplementary Table S2) containing plasmid (pDrive-601 NPS) as the template, the 601 NPS Fwd/Rev Entry-Exit–Cy5 and the 601 NPS Dyad–Cy5 DNA constructs were amplified by PCR (18 (link)). Similarly the 5S NPS Fwd Entry-Exit–Cy5 DNA construct was amplified by PCR from a plasmid (pBSII SK(-)-5S rDNA NPS) containing the Xenopus borealis 5S rDNA NPS sequence (Supplementary Table S2) (18 (link)). The reverse primers used in the PCRs were designed to incorporate biotin modified 78 base pair (bp) extensions to the 3′ end of each NPS (Supplementary Table S2 and Figure S1). After PCRs, the DNA constructs were purified by ion-exchange HPLC on a Gen-Pak Fax column (Waters).
10
Preparation of Labeled G-Quadruplex DNA
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For bulk experiments, GQ DNA sequences and their complements were purchased unmodified from Integrated DNA Technologies (IDT). Single stranded DNA studies were conducted with an 18 mer ssDNA on the 5′ end, while duplexed DNA studies contained two separate 18 mer on both the 3′ and 5′ ends. For single molecule experiments, the same sequences as above were purchased containing an amine-modified thymine located 3 or 4 bases from the GQ-forming region. Constructs were labeled by incubating 10 mM CY3 or Cy5-NHS ester (GE Lifesciences) with .1 mM DNA in 100 mM sodium bicarbonate pH 8.5 buffer for 4–5 h. Excess dye was removed through two rounds of ethanol precipitation. Sequences were diluted in a standard G-quadruplex formation buffer: 20 mM Tris-HCl pH 7.5, 100 mM KCl.
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