Sulfo cy5 nhs

Manufactured by Lumiprobe

Sourced in United States

Sulfo-Cy5-NHS is a water-soluble NHS ester of the cyanine dye Cy5. It is used for the covalent labeling of proteins, peptides, and other biomolecules that contain primary amine groups.

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Lab products found in correlation

Pd minitrap g 25, by GE Healthcare (2 mentions) Nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions) Hoechst, by Thermo Fisher Scientific (1 mentions) Tcs sp2, by Leica camera (1 mentions) Phalloidin ifluor 488, by Abcam (1 mentions) Prolong gold antifade reagent mounting medium, by Thermo Fisher Scientific (1 mentions) Tcs sp2 confocal microscope, by Leica camera (1 mentions) Histrap hp column, by Cytiva (1 mentions) Akta basic fplc, by GE Healthcare (1 mentions) Kta explorer chromatography system, by GE Healthcare (1 mentions) Axiocam mr3, by Zeiss (1 mentions) Axio observer d1, by Zeiss (1 mentions) P scn bn nota, by Macrocyclics (1 mentions) Tcs sp8, by Leica camera (1 mentions) Hitrap q hp, by GE Healthcare (1 mentions)

13 protocols using sulfo cy5 nhs

Cell Surface Labeling with HATRIC-Cy3

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HATRIC was pre-coupled to equimolar amine-Cy3 (Lumiprobe) in 25 mM HEPES (pH 8.2) for 1.5 h at RT and 300 rpm in the dark. MDA-MB-231 cells cultured on coverslips were oxidized with 1 ml of 1.5 mM sodium periodate in PBS, pH 6.5 for 15 min and labeled with 6 µM HATRIC-Cy3 or amine-Cy3 (Control w/o HATRIC) in 1 ml PBS with 5 mM 5-MA (pH 7.4) for 1.5 h at 4 °C shaking in the dark. As a cell surface marker, cells were labeled with 0.5 ml 1 mM sulfo-NHS-Cy5 (Lumiprobe). Nuclei were stained with 0.5 ml 1 µg ml⁻¹ Hoechst (Molecular Probes H1399) for 10 min at 4 °C. Cells were fixed with 4% paraformaldehyde for 10 min at RT, mounted with anti-fade mounting medium (Molecular Probes Prolong Gold Antifade reagent P36934) and analyzed by confocal microscopy (Leica TCS SP2). For a permeabilized control, cells were first stained with sulfo-NHS-Cy5 and fixed, and then permeabilized with 0.1% Triton X-100 for 10 min at RT, before oxidation and labeling with HATRIC-Cy3.

Sobotzki N., Schafroth M.A., Rudnicka A., Koetemann A., Marty F., Goetze S., Yamauchi Y., Carreira E.M, & Wollscheid B. (2018). HATRIC-based identification of receptors for orphan ligands. Nature Communications, 9, 1519.

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Evaluating Filter-Grown Cell Cultures

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To evaluate the tightness of filter-grown cell cultures, cells were labeled at the apical or basolateral surface using 1 mM sulfo-NHS-Cy5 (Lumiprobe) in PBS, pH 8.0 for 15 min on ice in the dark and washed three times with PBS before fixation. For all microscopy samples, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.1% TritonX-100 in blocking buffer (1% FBS, 1% BSA in PBS with 0.02% sodium azide) for 10 min at room temperature. Samples were blocked for 1 h at room temperature or overnight at 4 °C in a blocking buffer. Cells were subsequently incubated with mouse anti-ZO1-AF555 antibody (Thermo Invitrogen MA3-39100-A555, 1:100) for 1 h with shaking. Nuclei were stained with 1 µg/mL Hoechst (Molecular probes H1399), and F-actin was stained with phalloidin-iFluor488 (Abcam, 1:1000) for 15 min at room temperature. Samples were fixed with 4% paraformaldehyde for 10 min at room temperature and mounted with Prolong Gold Antifade reagent mounting medium (Molecular Probes). Images were taken with a Leica TCS SP2 confocal microscope and processed using the FIJI software.

Koetemann A, & Wollscheid B. (2022). Apicobasal Surfaceome Architecture Encodes for Polarized Epithelial Functionality and Depends on Tumor Suppressor PTEN. International Journal of Molecular Sciences, 23(24), 16193.

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Purification and Labeling of E. coli RNA Polymerase

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E. coli RNA polymerase (RNAP) has been purified as has previously been
described.⁵² Briefly, the RNAP holoenzyme (no σ factor) was
overexpressed in E. coli BL21 (DE3), pelleted, and stored overnight at
−80 °C. Pellets were redissolved in lysis buffer and cells were lyzed using a
homogenizer. The supernatant was clarified twice using centrifugation and RNAP was
isolated using a HisTrap HP column (Cytiva). Fractions were analyzed on SDS-PAGE and
relevant fractions were pooled and purified using Heparin affinity chromatography on an
AKTA Basic FPLC (GE Healthcare). Fractions were analyzed on SDS-PAGE and relevant
fractions were combined. For labeling, RNAP was labeled using NHS-sulfoCy5 (Lumiprobe)
following supplier instructions. The reaction took place for 4 h at room temperature and
excess dye was removed through multiple rounds of dialysis. RNAP-Cy5 was stored in a 50
v/v % glycerol storage buffer at −20 °C. Before use, RNAP-Cy5 was dialyzed to
a glycerol-free working buffer, and the concentration was determined using a Bradford
assay.

Schoenmakers L.L., Yewdall N.A., Lu T., André A.A., Nelissen F.H., Spruijt E, & Huck W.T. (2023). In Vitro Transcription–Translation in an Artificial Biomolecular Condensate. ACS Synthetic Biology, 12(7), 2004-2014.

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Fluorescent Labeling of Plant Virus Nanoparticles

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PVX-Cy5 fluorescent particles were synthesized by coupling NHS-Sulfo-Cy5 or Maleimide-Sulfo-Cy5 (Lumiprobe) to PVX via lysine or cysteine residues, respectively. PVX (at 2 mg mL⁻¹) was reacted with 0.5 molar excess of sulfo-Cy5 dye/CP in 0.1 M potassium phosphate buffer (pH 7.0) supplemented with 10% (v/v) DMSO on a rotisserie overnight at room temperature. Dye labeled PVX was purified by ultracentrifugation at 112 000g for 3 hours over a 30% (w/v) sucrose cushion. The resulting pellet was resuspended in 0.1 M potassium phosphate buffer pH 7. Post-purification, PVX concentration and number of Cy5/PVX in the resulting PVX-Lys-Cy5 (PVX-Cy5) and PVX-Cys-Cy5 particles were determined by UV-vis spectroscopy using the Beer–Lambert law and the PVX and Sulfo-Cy5-specific extinction coefficients of 2.97 mL mg⁻¹ cm⁻¹ at 260 nm and 271 000 M⁻¹ cm⁻¹ at 647 nm, respectively. Particle integrity was verified using size exclusion chromatography using a Superose6 column on the ÄKTA Explorer chromatography system (GE Healthcare).

Shukla S., Roe A.J., Liu R., Veliz F.A., Commandeur U., Wald D.N, & Steinmetz N.F. (2020). Affinity of plant viral nanoparticle potato virus X (PVX) towards malignant B cells enables cancer drug delivery. Biomaterials science, 8(14), 3935-3943.

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Fluorescent Labeling of Viral Capsids

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CPMV-Cy5, CCMV-Cy5, SeMV-Cy5 and PhMV-Cy5 formulations were synthesized by conjugating N-hydroxysuccinimide-activated esters of Sulfo-Cy5 (NHS-Sulfo-Cy5, Lumiprobe, Hunt Valley, MD) to surface exposed lysine residues. Briefly, Sulfo-Cy5 conjugations were performed on CPMV, CCMV, SeMV and PhMV using dye/lysine ratios of 1, 1/6, 2 and 1/2, respectively. Bioconjugation was carried out overnight in 0.1 M sodium phosphate buffer (KP, pH 7.4) with of 10% (v/v) DMSO and at 2 mg mL⁻¹ protein concentration. Dye-labeled particles were purified by ultracentrifugation (112 000 g, 1 h). The pellet was resuspended in 0.1 M KP buffer and protein concentration was determined using BCA assay. Dye numbers were determined through UV-vis analysis using the molar extinction coeffecient ε_sui_fo-Cy5 = 271 000 L mol⁻¹ cm⁻¹ at 647 nm. Dye conjugation to viral coat proteins was also confirmed using SDS-PAGE as described above and fluorescent bands were visualized on an AlphaImage gel documentation system (protein simple) using a 607 nm excitation.

Shukla S., Wang C., Beiss V., Cai H., Washington T I.I., Murray A.A., Gong X., Zhao Z., Masarapu H., Zlotnick A., Fiering S, & Steinmetz N.F. (2020). The unique potency of Cowpea mosaic virus (CPMV) in situ cancer vaccine. Biomaterials science, 8(19), 5489-5503.

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Fluorescent Labeling of Cowpea Mosaic Virus

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CPMV was propagated in black-eyed pea No. 5 plants (Vigna unguiculata) by mechanical inoculation and purified as reported in previous work.⁸⁸N-Hydroxysuccinimide chemistry was used to conjugate fiuorescent dye cyanine-5 succinimide ester (NHS-sulfo-Cy5; Lumiprobe) to CPMV’s surface lysine residues to obtain Cy5-CPMV. Briefly, 900 molar excess of NHS-sulfo-Cy5 was added to the CPMV in 10 mM potassium phosphate (KP) buffer (2 mg·mL⁻¹) and mixed at room temperature for 2 h, protected from light.⁵⁰ The solution was ultracentrifuged at 4 °C at 52000g for 1 h over a 30% (w/v) sucrose cushion. The resulting pellet was resuspended in 10 mM KP and stored at 4 °C until further characterization.

Affonso de Oliveira J.F., Chan S.K., Omole A.O., Agrawal V, & Steinmetz N.F. (2022). In Vivo Fate of Cowpea Mosaic Virus In Situ Vaccine: Biodistribution and Clearance. ACS nano, 16(11), 18315-18328.

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Fluorescent Labeling of CPMV Nanoparticles

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CPMV was labelled with sulfo-Cy5-NHS (Lumiprobe) targeting its solvent-exposed surface lysine residues (CPMV displays a total of 300 solvent-exposed surface lysine side chains per particle^{22 (link)}). The reaction was carried out using 5 equivalents of sulfo-Cy5-NHS per coat protein in 0.1 M KP buffer (pH 7.0) at room temperature overnight, with agitation. The reaction mixture was then centrifuged at 112000g for 1 h to remove excess dyes, and resuspended in 0.1 M KP buffer (pH 7.0) overnight. Further purification to remove potential aggregates involved centrifugation at 13000g for 10 min. CPMV–Cy5 was eluted using PD Minitrap G-25 desalting columns (GE Healthcare) to remove excess free Cy5 dyes.

Chariou P.L., Beiss V., Ma Y, & Steinmetz N.F. (2021). In situ vaccine application of inactivated CPMV nanoparticles for cancer immunotherapy. Materials Advances, 2(5), 1644-1656.

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Sulfo-Cy5 Labeling of TnaA and FMO Proteins

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A few grains of sulfo-Cy5-NHS (Lumiprobe) were dissolved in ddH₂O. The concentration was determined using the absorption at 640 nm, ε = 271,000 cm⁻¹ M⁻¹ using an ND-1000 spectrophotometer (ThermoScientific). The reagent was added in an equimolar ratio to 60 µM of TnaA or FMO protein (~1:30 ratio of NHS to lysine residues for both enzymes) in PBS and left for 1 h at 4 °C. Unreacted dye was removed using a PD-25 spin trap column (GE Healthcare). The average labeling per protein was measured using the absorption of the dye at 640 nm and at 280 nm for the protein, TnaA-Cy5 degree of labeling: 0.48, FMO-Cy5 degree of labeling: 0.56. For the experiments regarding the uptake without Ni²⁺ present, the degree of labeling was TnaA-Cy5: 0.52, FMO-Cy5: 0.32. The loading and imaging of these labeled proteins were performed as described above. For excitation of the Cy5 dye a 633 nm He/Ne laser was used. The brightfield images were made with a Zeiss Axio Observer D1 equipped with an LD Plan-Neofluar 40×/0.6 Corr, Halogen lamp, and an AxioCamMR3.

Altenburg W.J., Yewdall N.A., Vervoort D.F., van Stevendaal M.H., Mason A.F, & van Hest J.C. (2020). Programmed spatial organization of biomacromolecules into discrete, coacervate-based protocells. Nature Communications, 11, 6282.

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Fluorescent Labeling of Viral Capsids

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CPMV comprises 180 coat proteins and displays a total of 300 surface-exposed lysine side chains³⁰. PhMV also comprises 180 identical coat proteins, but each displays four surface-exposed lysine side chains to make 720 in total^{31 (link)}. CPMV and PhMV were labelled with sulfo-Cy5-NHS (Lumiprobe) using N-hydroxysuccinimide-activated esters that target the surface lysine residues. The reactions were carried out with a 1,200-fold (CPMV) or 900-fold (PhMV) molar excess of sulfo-Cy5-NHS in a 10 mM KP buffer (pH 7.0) at room temperature overnight, with agitation.

Chariou P.L., Dogan A.B., Welsh A.G., Saidel G.M., Baskaran H, & Steinmetz N.F. (2019). Soil mobility of synthetic and virus-based model nanopesticides. Nature nanotechnology, 14(7), 712-718.

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Labeling scFvs with Cy5 and NOTA

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scFvs (3B4 and M4) were reacted with 25-fold molar excess sulfo-Cy5-NHS (Lumiprobe) in 0.1 M NaHCO₃ (pH ~9) at 4°C overnight. The scFv conjugates were purified by size exclusion chromatography (SEC) (P-10, GE Healthcare) using PBS as an eluent. The pure fractions of scFv-Cy5 were confirmed using SEC column chromatography by monitoring peaks at OD214 nm and OD646 nm. The concentration of sulfo-Cy5 of scFv conjugates was calculated by measuring absorption at 646 nm (ε = 271,000 M^-1cm^-1). The metal chelator, p-SCN-Bn-NOTA [S-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid] (Macrocyclics), was conjugated with scFvs in a similar reaction condition used for the optical dye conjugation reaction; 0.1 M ammonium acetate (pH 6.5) was used for SEC (P-10, GE Healthcare) buffer exchange. NOTA-conjugated scFv antibodies were analyzed by MALDI-TOF mass spectrometry (Voyager), and sinapinic acid was used as a matrix. Protein concentration was measured using BCA protein assay, and the protein purity and size were confirmed by SDS-PAGE and SEC.

Kim H.Y., Wang X., Kang R., Tang D., Boone B.A., Zeh HJ I.I.I., Lotze M.T, & Edwards W.B. (2018). RAGE-specific single chain Fv for PET imaging of pancreatic cancer. PLoS ONE, 13(3), e0192821.

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