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Dylight 488 nhs ester

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DyLight 488 NHS Ester is a fluorescent labeling reagent used for the covalent attachment of fluorescent dyes to proteins and other biomolecules. It contains the NHS ester functional group that reacts with primary amines to form a stable amide bond.

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Lab products found in correlation

Lysotracker deep red, by Thermo Fisher Scientific (3 mentions) Sulforhodamine b, by Merck Group (3 mentions) Trastuzumab, by Roche (3 mentions) T dm1, by Roche (3 mentions) Dylight650 nhs ester, by Thermo Fisher Scientific (2 mentions) Cr3022, by Absolute Antibody (2 mentions) Propidium iodide, by Merck Group (2 mentions) Antibody against β tubulin, by Merck Group (2 mentions) 1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride edc, by Tokyo Chemical Industry (1 mentions) D pbs, by Fujifilm (1 mentions) Paraformaldehyde (pfa), by Fujifilm (1 mentions) Blocking one, by Nacalai Tesque (1 mentions) 3 o methyl d glucose, by Merck Group (1 mentions) L fucose, by AppliChem (1 mentions) Methyl α l fucopyranoside, by Biosynth (1 mentions) D glucose, by Biosynth (1 mentions) D galactose, by Biosynth (1 mentions) μ slide, by Ibidi (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Fv1000 confocal microscope, by Olympus (1 mentions)

Spelling variants of this product

DyLight 488 (158 citations) DyLight 488 N‐hydroxysuccinimide (NHS) ester (3 citations) NHS-esters (2 citations)

28 protocols using dylight 488 nhs ester

1

Synthesis and Modification of PEG Polymers

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Alpha-methoxy-ω-amino PEG (MeO-PEG-NH2; Mn = 2400; Mw/Mn = 1.11; NOF, Tokyo, Japan) was purified using an ion-exchange CM Sephadex C-50 column (GE Healthcare, Buckinghamshire, UK) before use. Alpha-acetal-ω-amino PEG (acetal-PEG-NH2) was synthesized as described previously [27 (link)]. Beta-benzyl-l-aspartate N-carboxy-anhydride (BLA-NCA; NOF), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC; Tokyo Chemical Industry, Tokyo, Japan), Cyclo[RGDfk(CX-)] (cRGD peptide, X = 6-aminocaproic acid, ∊-Acp; Peptide Institute, Osaka, Japan), sulfo-Cy3 mono-reactive dye (Lumiprobe, Orlando, Florida, USA), sulfo-Cy5 mono-reactive dye (Lumiprobe), DyLight488 NHS ester (Thermo Fischer Scientific, Waltham, Massachusetts, USA), methylamine solution (Me–NH2; 40%; Wako Pure Chemical Industries, Osaka, Japan), sodium hydroxide (NaOH; Koso Chemical, Tokyo, Japan), hydrochloric acid (HCl; Koso Chemical), acetic acid (CH3COOH; Nacalai Tesque, Tokyo, Japan), ferucarbotran (SPIO) solution (Resovist®, Fujifilm RI Pharma, Tokyo, Japan), Blocking one (Nacalai Tesque), 4% paraformaldehyde (4% PFA; Wako), Hoechst 33342 (Hoechst; Dojindo Laboratories, Kumamoto, Japan), and other reagents were used without further purification. PBS-Tween solution (0.1% v/v PBS-T) was prepared from Dulbecco’s phosphate-buffered saline (D-PBS; Wako) and Tween 20 solution (10% w/v, Wako).
Kawamura W., Miura Y., Kokuryo D., Toh K., Yamada N., Nomoto T., Matsumoto Y., Sueyoshi D., Liu X., Aoki I., Kano M.R., Nishiyama N., Saga T., Kishimura A, & Kataoka K. (2015). Density-tunable conjugation of cyclic RGD ligands with polyion complex vesicles for the neovascular imaging of orthotopic glioblastomas. Science and Technology of Advanced Materials, 16(3), 035004.
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2

Fluorescent Labeling of Monoclonal Antibody

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Monoclonal antibody CR3022 was purchased from Absolute Antibody (catalog number Ab01680–10.0). Biotin-PEG4-Amine (Nanocs, catalog number B-P4A-1) was dissolved in distilled water at a concentration of 10 mM (4.36 mg/ml). Fifty micrograms of DyLight650 NHS Ester (Thermo Fisher, catalog number 62266, MW = 1066) was dissolved in 100 μl phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4), resulting in a 0.47 mM solution. Fifty micrograms of DyLight488 NHS Ester (Thermo Fisher, catalog number 46403, MW = 1011) was dissolved in 100 μl PBS, resulting in a 0.47 mM solution. Eighty microliters of the DyLight650 NHS Ester or DyLight488 NHS Ester solution were mixed with 2.4 μl of the biotin-PEG4-Amine stock solution, corresponding to a dye:amine ratio of 1.57:1. After 1 h at room temperature, the reaction was quenched by the addition of 1 μl of 2 M Tris-HCl pH 8.0. We assumed that the reaction was complete, resulting in a 0.288 mM solution of biotin-PEG4-dye. These solutions were used without further purification, because the unreacted dye is not expected to interfere with the assay, as it is removed in multiple washing steps in the MBBA procedure.
Hattori T., Koide A., Panchenko T., Romero L.A., Teng K.W., Corrado A.D, & Koide S. (2020). Multiplex bead binding assays using off-the-shelf components and common flow cytometers. Journal of Immunological Methods, 490, 112952.
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3

Glycan Synthesis and Characterization Protocol

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l-fucose was purchased from AppliChem (Darmstadt, Germany). Methyl α-l-fucopyranoside, d-glucose, and d-galactose were purchased from Carbosynth (Compton, UK). 3-O-methyl-d-glucose, biotin, and streptavidin were purchased from Sigma-Aldrich (St. Louis, MO, USA). biotinylated α-l-fucoside was purchased from Synthaur LLC (Moscow, Russia). Methylated disaccharide 3,6-O-Me2-d-Glcβ1-4(2,3-O-Me2)-l-Rhaα-O-(p-C6H4)-O-CH2CH2NH2 was synthesized as described previously [29 (link)]. DyLight 488 NHS ester was purchased from ThermoScientific (Rockford, MI, USA). Protein molecular Marker III was purchased from AppliChem (Darmstadt, Germany). Other chemicals were purchased from Sigma-Aldrich, Duchefa, ForMedium and Applichem companies.
Faltinek L., Fujdiarová E., Melicher F., Houser J., Kašáková M., Kondakov N., Kononov L., Parkan K., Vidal S, & Wimmerová M. (2019). Lectin PLL3, a Novel Monomeric Member of the Seven-Bladed β-Propeller Lectin Family. Molecules, 24(24), 4540.
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4

Fluorescent labeling of LDL for cellular uptake

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LDL with a density of 1.019 to 1.063 g/mL was isolated from plasma of a healthy, normolipidemic donor through gradient ultracentrifugation after which it was fluorescently labeled with DyLight 488 NHS-Ester (ThermoFisher Scientific) for 1 hour according to the manufacturer’s protocol and dialyzed against PBS overnight.42 (link)After 24 hours of coculture (HepG2 and PBMCs or HepG2 and B-cell precursor acute lymphoblastic leukemia cells), 4 µg DyLight apoB-labeled LDL per well was added. Thirty minutes later, HepG2 cells were washed twice with ice-cold PBS +0.2% BSA after which they were lysed on ice for 30 minutes with ice-cold RIPA buffer (Pierce, Rockford, IL) supplemented with protease inhibitors (Complete; Roche). The lysates were centrifuged at 13 523g for 15 minutes at 4°C. The fluorescence at 488 nm in the supernatant was determined and compared with cells that were not incubated with labeled LDL.
Loaiza N., Hartgers M.L., Reeskamp L.F., Balder J.W., Rimbert A., Bazioti V., Wolters J.C., Winkelmeijer M., Jansen H.P., Dallinga-Thie G.M., Volta A., Huijkman N., Smit M., Kloosterhuis N., Koster M., Svendsen A.F., van de Sluis B., Hovingh G.K., Grefhorst A, & Kuivenhoven J.A. (2020). Taking One Step Back in Familial Hypercholesterolemia: STAP1 Does Not Alter Plasma LDL (Low-Density Lipoprotein) Cholesterol in Mice and Humans. Arteriosclerosis, Thrombosis, and Vascular Biology, 40(4), 973-985.
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5

Glycan array screening of SL2-1 binding

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Purified SL2-1 was labeled with DyLight 488 NHS Ester (Thermo Scientific) according to the manufacturer’s instructions and dialyzed against 20 mM Tris-HCl, pH 7.4, 150 mM NaCl using 10,000 MWCO dialysis membrane. DyLight 488 labeled protein was used for glycan array screening at the Protein-Glycan Interaction Core H of the Consortium for Functional Glycomics (Emory University, Atlanta, GA).
The specificity of SL2-1 was determined by screening its binding on the printed array (version 5.2) consisting of 609 mammalian glycans. Detailed information about the structures and linkers of these glycans can be found at https://glycopattern.emory.edu. The printed array was probed with 200 μg/mL of SL2-1 diluted in 20 mM Tris-HCL pH 7.4, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.05% Tween 20, 1% BSA, in 6 replicate binding experiments. The highest and lowest point from each set of six replicates was discarded, and the average RFU value of 4 replicates, the standard deviation, and % CV (% CV = 100 * Standard Deviation/Mean) was calculated.
Vainauskas S., Duke R.M., McFarland J., McClung C., Ruse C, & Taron C.H. (2016). Profiling of core fucosylated N-glycans using a novel bacterial lectin that specifically recognizes α1,6 fucosylated chitobiose. Scientific Reports, 6, 34195.
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6

Fluorescent Labeling and Imaging of Prostate Cancer Cells

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The NBs were labeled at a 1:3 molar
ratio with Dylight 488 NHS-ester (Thermo Scientific, IL). Phycoerythrin
(PE)-anti PSMA antibody (BioLegend, CA) and Hoechst 33342 (Invitrogen)
were incubated for 15 min with 3 × 104 PC3-PIP or
PC3-flu cells, which were grown overnight in an 8-well μ-slide
(ibidi GmbH, Germany) in the presence or absence of 100 nM labeled
NB. NB7cys and NB7cysDOX were labeled at a 1:3 molar ratio with Dylight
650 NHS-ester. Hoechst 33342 and 1.5 μg/mL DOX (Teva, Israel)
or an equivalent molar amount of labeled NB7cys or labeled NB7cysDOX
were incubated with PC3-PIP and PC3-flu cells, grown as described
above. The cells were imaged with an Olympus FV1000 confocal microscope
(Olympus, Japan), with a long-working distance ×60/1.35 numerical
aperture, oil-immersion objective.
Rosenfeld L., Sananes A., Zur Y., Cohen S., Dhara K., Gelkop S., Ben Zeev E., Shahar A., Lobel L., Akabayov B., Arbely E, & Papo N. (2020). Nanobodies Targeting Prostate-Specific Membrane Antigen for the Imaging and Therapy of Prostate Cancer. Journal of Medicinal Chemistry, 63(14), 7601-7615.
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7

Investigating Antibody-Drug Conjugate Mechanisms

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Both T‐DM1 and trastuzumab were purchased from F. Hoffmann‐La Roche (Basel, Switzerland). DM1 was provided by Jiangsu Hengrui Pharmaceutical Co. (Lianyungang, China). Bafilomycin A1 was obtained from Selleck Chemicals (Houston, TX, USA). DyLight 488 NHS Ester and LysoTracker Deep Red were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Propidium iodide, sulforhodamine B, and DAPI were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Acridine orange was purchased from China National Pharmaceutical Industry Corp (Beijing, China). Hertuzumab‐vc‐MMAE was obtained from Rongchang Pharmaceuticals, Ltd (Yantai, China).
Antibodies against HER2, GAPDH, and PARP were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody specific for β‐tubulin was purchased from Sigma‐Aldrich. The antibody against β‐actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alexa Fluor 488‐conjugated goat anti‐mouse IgG was purchased from Invitrogen (Carlsbad, CA, USA).
Wang H., Wang W., Xu Y., Yang Y., Chen X., Quan H, & Lou L. (2017). Aberrant intracellular metabolism of T‐DM1 confers T‐DM1 resistance in human epidermal growth factor receptor 2‐positive gastric cancer cells. Cancer Science, 108(7), 1458-1468.
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8

Tau Condensates FRAP Imaging

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Tau:suramin condensates containing 2% DyLight-488 labeled Tau (labeled using amine-reactive DyLight488-NHS ester (Thermo Scientific) following manufacturer instructions) were imaged before and directly after bleaching with a 488 nm laser (90% intensity; 6 loops). The recovery of the fluorescence in the bleached region (circular ROIs, diameter 1 to 2 µm), a similar non-bleached reference-ROI (inside a different condensate) and a background ROI (Region of interest) were monitored in parallel for 40 s. FRAP curves were background corrected and normalized to the background corrected reference signal. Experiments were performed at room temperature on a spinning disk confocal microscope (Eclipse-Ti CSU-X, Nikon) using a 60x oil objective (Fig. 1g).
Prince P.R., Hochmair J., Brognaro H., Gevorgyan S., Franck M., Schubert R., Lorenzen K., Yazici S., Mandelkow E., Wegmann S, & Betzel C. (2023). Initiation and modulation of Tau protein phase separation by the drug suramin. Scientific Reports, 13, 3963.
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9

Palmitoylation Impacts GAD65 Function

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rhGAD65 was depalmitoylated overnight by treatment with 200 mmol/L hydroxylamine (HA) and labeled with DyLight 488 NHS Ester (Thermo Fisher Scientific). Palmitoylated or depalmitoylated GAD65-488 was incubated at 10 µg/mL with 50,000 Priess cells/well in 96-well plates. Cells were stained with LIVE/DEAD Aqua (Molecular Probes) and analyzed by Cyan Flow Cytometer (Beckman Coulter) and FlowJo software.
Phelps E.A., Cianciaruso C., Michael I.P., Pasquier M., Kanaani J., Nano R., Lavallard V., Billestrup N., Hubbell J.A, & Baekkeskov S. (2016). Aberrant Accumulation of the Diabetes Autoantigen GAD65 in Golgi Membranes in Conditions of ER Stress and Autoimmunity. Diabetes, 65(9), 2686-2699.
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10

Phase Separation of TDP-43 Mutants

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Differential interference contrast (DIC) images and FRAP of WT TDP-43267–414 and mutant variants were conducted in 20 mM MES pH 6.1 with 150 mM NaCl or 15 µg/ml torula yeast RNA extract (WT DIC only). Control samples were prepared by omitting NaCl. Protein samples were then spotted onto glass coverslips. For fluorescence images and FRAP of TDP-43 C-terminal domain phase separation and fusion events, the N-terminal primary amine of WT TDP-43267–414 was conjugated to DyLight 488 NHS ester (Thermo Scientific). Excess unconjugated dye was removed by desalting through two 0.5 ml Zeba Spin Desalting columns. Fluorescently labeled TDP-43267–414 was added at a final concentration of 5 nM to 50 µM unlabeled TDP-43267–414 in the presence of 150 mM NaCl as described above.
Conicella A.E., Zerze G.H., Mittal J, & Fawzi N.L. (2016). ALS mutations disrupt phase separation mediated by -helical structure in the TDP-43 low complexity C-terminal domain. Structure (London, England : 1993), 24(9), 1537-1549.
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