Cy3b nhs ester

Manufactured by GE Healthcare

Cy3B-NHS ester is a fluorescent labeling reagent used for the covalent attachment of dyes to biomolecules, such as proteins, peptides, and nucleic acids. It provides efficient labeling with a bright, stable, and pH-insensitive fluorescent signal.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

NHS-Cy3B (4 citations)

12 protocols using cy3b nhs ester

Labeling SNAP-Tagged PUF Proteins with Cy3B Dye

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cy3B-labeled SNAP tag substrate was prepared by coupling Cy3B NHS
ester (GE Healthcare, 0.75 μmol) with 1.5-fold excess(1.13
μmol) of amine-terminated benzylguanine (NH2-BG; New England BioLabs)
in the presence of 1.13 μmol triethylamine in dimethylformamide. The
reaction (103 μL) was incubated overnight on a rotating platform at
30°C. The Cy3B-BG product was purified by reverse phase HPLC on an
Agilent ZORBAX Eclipse Plus 95Å column and dried by speed-vac
evaporation (46% yield).
SNAP-tagged PUF proteins were labeled by incubating 5–10
μM of purified protein with 20 μM of Cy3B-BG in Buffer C. The
tube was covered with aluminum foil and rotated at 4°C for
12–14 h. Unincorporated dye was removed with Zeba Spin Desalting
Columns (Thermo Fisher Scientific) equilibrated with Buffer C; the protein
was concentrated using Amicon Ultra 10KDa filters and diluted two-fold with
Buffer C containing 80% glycerol for final storage at −20°C.
The labeling efficiencies (based on total protein concentration and Cy3B
absorbance at 559 nm; Cy3B extinction coefficient: 130,000
M⁻¹cm⁻¹) were 60% (PUM2-SNAP), 53%
(SNAP-PUM1) and 36% (mutant SNAP-PUM1).

Jarmoskaite I., Denny S.K., Vaidyanathan P.P., Becker W.R., Andreasson J.O., Layton C.J., Kappel K., Shivashankar V., Sreenivasan R., Das R., Greenleaf W.J, & Herschlag D. (2019). A Quantitative and Predictive Model for RNA Binding by Human Pumilio Proteins. Molecular cell, 74(5), 966-981.e18.

+ Open protocol

+ Expand

Readout Probe Design for seqFISH+

Cited 3 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

Readout probes of 12–15-nt in length were designed for seqFISH as previously described^{20 (link),21 (link)}. In brief, a set of probe sequences was randomly generated with combinations of A, T, G or C nucleotides with a GC-content range of 40–60%. To minimize cross-hybridization between the readout probes, any probes with ten or more contiguously matching sequences between the readout probes were removed. The readout probes for sequential immunofluorescence were similarly designed except ‘C’ nucleotide is omitted^{60 (link)}. The 5’ amine-modified DNA oligonucleotides (Integrated DNA Technologies) with the readout probe sequences were conjugated in-house to Alexa Fluor 647-NHS ester (Invitrogen A20006) or Cy3B-NHS ester (GE Healthcare PA63101) or Alexa Fluor 488-NHS (Invitrogen A20000) as described before^{20 (link),21 (link)}, or fluorophore conjugated DNA oligonucleotides were purchased from Integrated DNA Technologies. In total, 240 unique readout probes^{21 (link)} were designed and synthesized for DNA seqFISH+ experiments, and subsets of those readout probes were used for RNA seqFISH experiments. The cost for 240 readout probes for DNA seqFISH+ were approximately $15,000 with 5’ amine-modified DNA oligonucleotides and dye conjugation in-house, and $50,000 with fully labeled purchase, which can be used over hundreds or thousands of experiments.

Takei Y., Yun J., Zheng S., Ollikainen N., Pierson N., White J., Shah S., Thomassie J., Suo S., Eng C.H., Guttman M., Yuan G.C, & Cai L. (2021). Integrated spatial genomics reveals global architecture of single nuclei. Nature, 590(7845), 344-350.

+ Open protocol

+ Expand

Multiplexed Spatial Transcriptomics via DNA seqFISH+

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

To implement the two-layer DNA seqFISH+ strategy, 96 unique readout probes were used in each fluorescent channel for a total of 288 unique readout probes for 3 fluorescent channels. The readouts probe sequences were obtained from our previous DNA seqFISH+ studies^{7 (link),8 (link)} as well as additional orthogonal readout probe sequences were generated and validated similarly to those previous studies. The RNA seqFISH+ readout probe sequences were selected from a subset of the two-layer DNA seqFISH+ readout probe sequences. The readout probe sequences (12-15-nt) for sequential immunofluorescence were selected from our previous studies^{7 (link),8 (link)} and further designed and validated with the same criteria for this study. The fluorescently-labeled readout probes (Integrated DNA Technologies) that can bind to the readout sequences on the primary probes or primary antibodies were conjugated in-house to Alexa Fluor 647–NHS ester (Invitrogen A20006), Cy3B–NHS ester (GE Healthcare PA63101), or Alexa Fluor 488–NHS ester (Invitrogen A20000) as described before^{17 (link)} or directly purchased (Integrated DNA Technologies).

Takei Y., Yang Y., White J., Yun J., Prasad M., Ombelets L.J., Schindler S, & Cai L. (2023). High-resolution spatial multi-omics reveals cell-type specific nuclear compartments. bioRxiv.

+ Open protocol

+ Expand

Fluorescently Labeled Molecular Beacons for RNA Imaging

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

MTRIP design and synthesis is described in detail elsewhere^{46 (link)}. Briefly, 2′-O-methyl RNA/DNA chimeric nucleic acid oligonucleotides targeting the RSV A2 genome contain a 5′-biotin modification (Biosearch Technologies) and 5 internal dT amino groups. These ligands were then labeled with either Cy3B NHS ester (GE Healthcare) or DyLight 650 NHS ester (Pierce) according to manufacturer instructions. Unbound dye was removed via centrifugation in a 3 kDa spin column (Millipore) and stored at −20 °C until use. Probes were assembled by mixing labeled oligonucleotides with Neutravidin (Pierce) at a 5:1 molar ration and allowed to react for 1 h at room temperature. Unbound oligonucleotides were filtered out by centrifugation through a 30 kDa spin column (Millipore).
MTRIPs were delivered to cells at 30 nM concentration in Opti-MEM I medium (Invitrogen) containing 0.2 U/mL activated SLO (Sigma) after washing the cells with DPBS without Ca²⁺ and Mg²⁺. The membrane was then allowed to recover in fresh growth medium for 15 min before imaging.

Vanover D., Smith D.V., Blanchard E.L., Alonas E., Kirschman J.L., Lifland A.W., Zurla C, & Santangelo P.J. (2017). RSV glycoprotein and genomic RNA dynamics reveal filament assembly prior to the plasma membrane. Nature Communications, 8, 667.

+ Open protocol

+ Expand

Labeling mRNA with Oligonucleotide Probes

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

We designed four 2’ O-methyl RNA-DNA chimeric oligonucleotides, each containing a 17–18 nucleotide binding region and 5–7 poly(T) linker^{28 (link)–30 (link)}. Oligos were complementary to 4 adjacent sequences across the mRNA 3’ UTR regions and contained 3–4 amino-modified thymidines each, as well as a 5’ biotin modification (Biosearch Technologies). Labeled oligos were synthesized by conjugating either Cy3B NHS ester (GE Healthcare) or DyLight 650 (Thermo Fisher) to the amine groups on the oligonucleotides followinhisg the manufacturer’s protocol. Complete MTRIPs were assembled by incubating the labeled oligos with Neutravidin (Pierce) for 1 h at RT followed by filtration using 30 kDa MWCO centrifugal filters (Millipore). mRNA was buffer exchanged into 1× PBS, heated to 70 °C for 10 min, and immediately placed on ice. Denatured mRNA was then combined with MTRIPs in a 1:1 ratio for each MTRIP and incubated at 37 °C overnight. Labeled mRNA was then buffer exchanged into water and concentrated using a 30 kDa filter before being injected into the animals.

Tiwari P.M., Vanover D., Lindsay K.E., Bawage S.S., Kirschman J.L., Bhosle S., Lifland A.W., Zurla C, & Santangelo P.J. (2018). Engineered mRNA-expressed antibodies prevent respiratory syncytial virus infection. Nature Communications, 9, 3999.

+ Open protocol

+ Expand

Monovalent mRNA-Targeting Reporter Probes

Check if the same lab product or an alternative is used in the 5 most similar protocols

Monovalent MTRIPs (mMTRIPS) consist of a NeutrAvidin core bound to an mRNA-targeting ligand via an aromatic hydrazine and aldehyde linkage (Hynic-4FB, Solulink) and to four biotinlated and fluorescently labeled reporter oligonucleotides. The mRNA-targeting ligand – containing a 5′ thiol modification (Biosearch Technologies) – are first conjugated to a 30x molar excess of maleimide HyNiC crosslinker (MHPH, Solulink). NeutrAvidin (Sigma) is labeled with 4FB groups (S-4FB, Solulink) to achieve a ratio of 2 4FB groups per molecule, as quantified by colorimetric reaction with 2-hydrazinopyridine (Solulink) by UV-Vis (Abs=350). The oligonucleotides and NeutrAvidin are separately filtered and buffer exchanged then combined according to the manufacturer protocol. The concentration of the resulting modified NeutrAvidin is quantified using a BCA Protein Assay Kit (Pierce). The reporter oligonucleotides contain a 5'-biotin modification and one dT-C6-NH₂ modification for conjugation with Cy3B-NHS ester (GE Healthcare) using manufacturer protocols. Free dye was removed using 3kD Nanosep spin columns (Pall Corp.). The purified ligands were resuspended in 1xPBS and mixed at a 5:1 molar ratio with the modified NeutrAvidin for 1 hour at RT. Free ligands were removed using 30 kD Nanosep spin columns.

Groves B., Chen Y.J., Zurla C., Pochekailov S., Kirschman J.L., Santangelo P.J, & Seelig G. (2015). Computing in mammalian cells with nucleic acid strand exchange. Nature nanotechnology, 11(3), 287-294.

+ Open protocol

+ Expand

Functionalized Surface Biomolecule Immobilization

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cy3B-NHS ester (PA63101) was acquired from GE Healthcare Life Sciences (Pittsburgh, PA). DNA was custom synthesized by Integrated DNA Technologies (Coralville, IA). Cyclo[Arg-Gly-Asp-d-Phe-Lys(PEG-PEG)] (PCI-3696-PI), elsewhere abbreviated as cRGD, was acquired from Peptides International (Louisville, KY). Streptavidin (S000–01) was obtained from Rockland-Inc (Pottstown, PA). μ-Slide VI^0.4 6-channel slides (80606) and 25 mm × 75 mm glass coverslips (10812) were purchased from Ibidi (Verona, WI). N-hydroxyl succinimide-5 kDa PEG-biotin (NHS-PEG-biotin, HE041024–5K) was purchased from Biochempeg (Watertown, MA). N-hydroxyl succinimide-5kDa mPEG (NHS-mPEG, PG1-SC-5k-1) was purchased from Nanocs (New York, NY). Sulfo-N-hydroxyl succinimide-acetate (sulfo-NHS-acetate, 26777) was purchased from Thermo-Fisher (Waltham, MA). (3-Aminopropyl)triethoxysilane (APTES, 440140, 99% purity) was purchased from Sigma-Aldrich. Tetraspek beads were purchased from Thermo-Fisher (T7279). Phalloidin-amine was purchased from Santa-Cruz biotechnology (sc-397330). All other reagents and materials (unless otherwise stated) were purchased from Sigma-Aldrich and used without purification. All buffers were prepared with 18.2 MΩ nanopure water.

Brockman J.M., Su H., Blanchard A.T., Duan Y., Meyer T., Quach M.E., Glazier R., Bazrafshan A., Bender R.L., Kellner A.V., Ogasawara H., Ma R., Schueder F., Petrich B.G., Jungmann R., Li R., Mattheyses A.L., Ke Y, & Salaita K. (2020). Live-cell super-resolved PAINT imaging of pN cellular traction forces. Nature methods, 17(10), 1018-1024.

+ Open protocol

+ Expand

Synthesis and Labeling of Fluorescent Biomolecules

Check if the same lab product or an alternative is used in the 5 most similar protocols

The double-stranded DNA oligonucleotide STD45T was prepared by automated synthesis (IBA GmbH). The sequence used was 5′TAAATCTAAAGTAACATAAGGTAACATAACGTAAGCTC-ATTCGCG-3′, where the underlined T base was labeled with Cy3b-NHS ester (GE Healthcare), as previously described (Crawford et al. 2013 (link)).
DNA Pol I and Klenow fragment (KF) were expressed, purified and fluorescently labeled as described (Joyce and Derbyshire 1995 (link); Joyce et al. 2008 (link); Santoso et al. 2010 (link)). Labelling efficiencies, quantified from UV–Vis spectra, were between 75 and 90 %. For electroporation, labeled proteins were dialysed into 50 mM Tris pH 7.4, 25 mM NaCl, 1 mM DTT, 50 % glycerol and stored at −20 °C.
C-terminal His₆-tagged ω [Cys68] was expressed using pET expression system and purified by Ni²⁺-affinity chromatography in denaturing conditions. ω [Cys68] was reduced either as described in (Kim et al. 2008 (link)) or using Reduce-Imm (Pierce) column as per manufacturer’s instructions. Cy3b-labeled ω [Cys68] was prepared using Cy3b maleimide dye (Amersham) as in (Kim et al. 2008 (link)). Cy3-ω [Cys68] was purified from unincorporated dye by Ni²⁺-affinity chromatography.

Sustarsic M., Plochowietz A., Aigrain L., Yuzenkova Y., Zenkin N, & Kapanidis A. (2014). Optimized delivery of fluorescently labeled proteins in live bacteria using electroporation. Histochemistry and Cell Biology, 142(1), 113-124.

+ Open protocol

+ Expand

Monovalent mRNA-Targeting Reporter Probes

Check if the same lab product or an alternative is used in the 5 most similar protocols

+ Open protocol

+ Expand

Conjugation of Fluorescent Dyes with BG-NH2

Check if the same lab product or an alternative is used in the 5 most similar protocols

Benzylguanine-conjugated Alexa Fluor 647 (#S9136S) and benzylguanine-conjugated Dyomics 649P1 (#S9159S) were purchased from New England BioLabs. Alexa Fluor 647 (#A20006), Alexa Fluor 680 (#A20008) and DyLight 650 (#62265) conjugated with succinimidyl ester (NHS ester) were purchased from Thermo Scientific. Atto 647N NHS ester (#18373) and Atto 655 NHS ester (#76245) were purchased from ATTO-TEC. Cy3B NHS ester (#PA63101), Cy5 NHS ester (#PA15101), and Cy5.5 NHS ester (#PA15601) were purchased from GE Healthcare. CF633 (#92133), CF647 (#92135), CF660C (#92137), and CF660R (#92134) conjugated with NHS ester were purchased from Biotium. Dyomics 654 conjugated with NHS ester (#654-01) was purchased from Dyomics (Jena). Each dye with NHS ester was reacted with BG-NH2 (#S9148S, New England Biolabs) in anhydrous dimethylformamide (DMF, #227056, Sigma-Aldrich) at 30 °C overnight according to the manufacturer's instructions (New England Biolabs).

Kim D.H., Chang Y., Park S., Jeong M.G., Kwon Y., Zhou K., Noh J., Choi Y.K., Hong T.M., Chang Y.T, & Ryu S.H. (2021). Blue-conversion of organic dyes produces artifacts in multicolor fluorescence imaging. Chemical Science, 12(25), 8660-8667.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!