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Alexa fluor 568 nhs ester

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Alexa Fluor 568 NHS Ester is a fluorescent dye used for labeling proteins and other biomolecules. It has an excitation maximum at 578 nm and an emission maximum at 603 nm, falling within the orange-red region of the visible spectrum. The NHS ester group allows the dye to covalently attach to primary amines on target molecules.

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Lab products found in correlation

Alexa fluor 488 nhs ester, by Thermo Fisher Scientific (3 mentions) Alexa fluor 647 nhs ester, by Thermo Fisher Scientific (2 mentions) Hydroxylamine, by Merck Group (1 mentions) Sudan black b, by Merck Group (1 mentions) Normal goat serum (ngs), by Vector Laboratories (1 mentions) Doxycycline, by Santa Cruz Biotechnology (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Zeba spin desalting columns 7k mwco 0.5 ml, by Thermo Fisher Scientific (1 mentions) Lsm 800, by Zeiss (1 mentions) 8 chamber slide, by Thermo Fisher Scientific (1 mentions) Exosome isolation reagent, by Thermo Fisher Scientific (1 mentions) C2 plus confocal microscope, by Nikon (1 mentions) Alexa fluor 647 maleimide, by Thermo Fisher Scientific (1 mentions) Sylgard 184 kit, by Dow (1 mentions) 1 2 dimyristoyl sn glycero 3 phospho l serine, by Avanti Polar Lipids (1 mentions) Carbon black nanopowder, by Merck Group (1 mentions) Thioflavin t ultrapure grade, by Eurogentec (1 mentions) Nhs fluorescein, by Thermo Fisher Scientific (1 mentions) Ab19348, by Abcam (1 mentions) Ab7120, by Abcam (1 mentions)

Close spelling variants of this product

Alexa Fluor 568 (1190 citations) Alexa 568 (337 citations) Alexa Fluors (21 citations) NHS-esters (2 citations)

14 protocols using alexa fluor 568 nhs ester

1

FRET-based Binding Assay for 14-3-3θ

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The Site 2 and Sites 2,4 peptides were fluorescently labeled at the amidated C-terminus. Both peptides were labeled with the donor probe Alexa Fluor 488 NHS Ester, whereas the Site 2 peptide was additionally labeled with the acceptor probe Alexa Fluor 568 NHS Ester (Thermo Fisher Scientific). The labeling reaction was performed at 25 °C for 1 h in 20 mM MES pH 6.8 and 50 mM NaCl and at a peptide concentration of 5 mg mL−1 with a 2-fold molar excess of fluorescent dye. The labeling reaction was stopped with the addition of a 10-fold molar excess of hydroxylamine (Sigma-Aldrich Corporation). Labeled peptides were separated from any remaining unlabeled peptide using a Symmetry300 C18 reverse phase HPLC column (Waters Corporation, Milford, MA). The peptides were lyophilized, resuspended in 100 mM HEPES pH 7.5, and dialyzed against 25 mM HEPES pH 7.5, 50 mM NaCl and 1 mM MgCl2. The donor-labeled peptides were pre-incubated with 14-3-3θ at a 1:1 molar ratio (i.e., one peptide per 14-3-3θ dimer). The acceptor-labeled Site 2 peptide (at 5 μM concentration) was then titrated into the preformed complexes. The steady-state fluorescence emission spectra were recorded at 25 °C, with an excitation wavelength of 495 nm (5-nm slit width) and an emission wavelength of 520 nm (5-nm slit width).
Kast D.J, & Dominguez R. (2019). Mechanism of IRSp53 inhibition by 14-3-3. Nature Communications, 10, 483.
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2

Alexa Fluor 568 Labeling of HDL

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Commercially purchased human HDL (Kalen Biomedical LLC) was labeled with Alexa Fluor 568 NHS Ester (ThermoFisher Scientific) following the manufacturer's protocol. Following conjugation, the reaction was dialyzed against 3x1L sterile 1x PBS over 3 days at 4°C using Slide-A-LyzerTM Dialysis Cassettes, 10K MWCO, 0.5 mL (ThermoFisher Scientific) where dialysis buffer was changed after each day. The efficiency of Alexa Fluor labeling was verified by running dialyzed Alexa Fluor labeled HDL (AF568-HDL) on an SDS-PAGE to compare the amount of Alexa Fluor labeled ApoA1 (approximately 25 kDa) to free Alexa Fluor. Additionally, to ensure the HDL particle remained intact after Alexa Fluor labeling, the size of AF568-HDL was compared with unlabeled HDL by running on an agarose gel followed by lipid staining with Sudan Black B (Sigma-Aldrich).
Fung K.Y., Wang C., Nyegaard S., Heit B., Fairn G.D, & Lee W.L. (2017). SR-BI Mediated Transcytosis of HDL in Brain Microvascular Endothelial Cells Is Independent of Caveolin, Clathrin, and PDZK1. Frontiers in Physiology, 8, 841.
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3

Hyaluronic Acid Visualization in Cells and Tissues

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Cells were plated at 5,000 cells/cm2 and subsequently induced with 0.2 μg/mL of doxycycline (Santa Cruz) for 24 h before being fixed with 4% paraformaldehyde. Antibodies and HABP were diluted 1:200 in 5% normal goat serum (Vector Laboratories) in PBS and incubated overnight at 4°C. For fluorescent HABP, HABP (Millipore Sigma) was labeled with Alexa Fluor 568 NHS Ester (Thermo Fisher Scientific) per the manufacturer’s protocol. Lectins were diluted to 1 μg/mL in 5% normal goat serum in PBS and incubated for 2 h at room temperature. For hyaluronic acid staining of cells and tissues, HABP was diluted to 0.125 μg/ml in 0.5% normal goat serum in PBS and incubated on samples for 24 h. Cell samples were imaged on a Zeiss LSM inverted 880 confocal microscope using a 40x water immersion objective (NA 1.1). In addition to HABP, NaBH4-treated tissues were stained with 1 μg/mL Hoechst for 10 min and imaged on a Zeiss 880 upright confocal microscope with a 40x water dipping lens. Unstained tissue collagen was visualized with second harmonic generation using non-descan detectors.
Shurer C.R., Kuo J.C., Monét Roberts L., Gandhi J.G., Colville M.J., Enoki T.A., Pan H., Su J., Noble J.M., Hollander M.J., O’Donnell J.P., Yin R., Pedram K., Möckl L., Kourkoutis L.F., Moerner W.E., Bertozzi C.R., Feigenson G.W., Reesink H.L, & Paszek M.J. (2019). Physical Principles of Membrane Shape Regulation by the Glycocalyx. Cell, 177(7), 1757-1770.e21.
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4

Fluorescent Protein Labeling Protocol

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Proteins were labelled using Alexa Fluor™ 488 or Alexa Fluor™ 568 NHS Ester (Succinimidyl Ester) (Thermo Fisher) in HDB. Dye and proteins were reacted at 2;1 molar ratio. Reaction mixtures were rotated for 60 min at room temperature, then labelled protein was separated from unconjugated dye using Zeba™ Spin Desalting Columns, 7K MWCO, 0.5 mL (ThermoFischer). Reaction and the column washing buffers were as same as protein elution buffer of Dynamin 1xA or Syndapin 1.
Imoto Y., Raychaudhuri S., Ma Y., Fenske P., Sandoval E., Itoh K., Blumrich E.M., Matsubayashi H.T., Mamer L., Zarebidaki F., Söhl-Kielczynski B., Trimbuch T., Nayak S., Iwasa J.H., Liu J., Wu B., Ha T., Inoue T., Jorgensen E.M., Cousin M.A., Rosenmund C, & Watanabe S. (2022). Dynamin is primed at endocytic sites for ultrafast endocytosis. Neuron, 110(17), 2815-2835.e13.
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5

Alexa Fluor 568 Labeling of Teratocytes

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For Alexa-NHS labeling, teratocytes were collected and cultured with water-soluble Alexa Fluor™ 568 NHS Ester (Thermo Fisher Scientific, Hudson, NH, USA) (1 μM) in TNM-FH medium with 10% FBS medium for 30 min. The medium was removed and the cells were washed with PBS three times followed by addition of fresh TNM-FH medium with exosome-depleted FBS and cultured for 6 h. The culture medium was then collected and subjected to exosome isolation using an exosome isolation reagent (Invitrogen). The Pxem_ZJU cells were adhered to an 8-chamber slide (Thermo Fisher Scientific) incubated with/without the fluorescently labeled medium for 18 h. DAPI was added during the last 5 min. Slides were analyzed by confocal microscopy (LSM 800, Zeiss, Jena, Germany).
Wang Z.Z., Ye X.Q., Shi M., Li F., Wang Z.H., Zhou Y.N., Gu Q.J., Wu X.T., Yin C.L., Guo D.H., Hu R.M., Hu N.N., Chen T., Zheng B.Y., Zou J.N., Zhan L.Q., Wei S.J., Wang Y.P., Huang J.H., Fang X.D., Strand M.R, & Chen X.X. (2018). Parasitic insect-derived miRNAs modulate host development. Nature Communications, 9, 2205.
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6

Cytodex3 Microcarrier Fluorescent Staining

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Cytodex3 microcarriers were stained with either Alexa Fluor 488 NHS Ester (Thermo Scientific, #A20000) or Alexa Fluor 568 NHS Ester (Thermo Scientific, #A20003) according to the manufacturer’s instructions.
Fischer A., Koopmans T., Ramesh P., Christ S., Strunz M., Wannemacher J., Aichler M., Feuchtinger A., Walch A., Ansari M., Theis F.J., Schorpp K., Hadian K., Neumann P.A., Schiller H.B, & Rinkevich Y. (2020). Post-surgical adhesions are triggered by calcium-dependent membrane bridges between mesothelial surfaces. Nature Communications, 11, 3068.
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7

Preparation and Imaging of Ni-NTA GUVs with Podocalyxin

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Giant Unilamellar Vesicles (GUVs) were prepared by electroformation as described previously (Angelova and Dimitrov, 1986 ). Briefly, lipids and dye dissolved in chloroform were spread on glass slides coated with ITO (Indium-Tin-Oxide). The slides were placed under vacuum for 2 h to remove all traces of organic solvents. The lipid films were hydrated and swelled in 120 mM sucrose at 55°C. GUVs were electroformed by the application of an oscillating potential of 1.4 V (peak-to-peak) and 12 Hz for 3 h (Busch et al., 2015 (link)). GUVs compositions were prepared with DOPC and increasing molar fractions of DOGS-Ni-NTA lipid (5, 10, 15, and 20 mol%). Bodipy-PC was used to label the lipids at a dye/lipid ratio of 1/2500. Recombinant His-tagged Podocalyxin was conjugated with Alexa Fluor 568 NHS Ester (Thermo Fisher Scientific), and the degree of labelling quantified according to the manufacturer’s protocol. GUVs were diluted in 20 mM HEPES, 50 mM NaCl, pH = 7.4 (120 mOsm) and then mixed with labeled Podocalyxin (~2 μM) for at least 20 minutes before imaging (GUVs/proteins = 1/1 by volume). GUVs were imaged on a Nikon C2plus confocal microscope using a 60x water immersion objective (NA 1.2). Lipids (Bodipy-PC) and protein (Alexa Fluor 568) were imaged through excitation at wavelength λ = 488 and 561 nm, respectively.
Shurer C.R., Kuo J.C., Monét Roberts L., Gandhi J.G., Colville M.J., Enoki T.A., Pan H., Su J., Noble J.M., Hollander M.J., O’Donnell J.P., Yin R., Pedram K., Möckl L., Kourkoutis L.F., Moerner W.E., Bertozzi C.R., Feigenson G.W., Reesink H.L, & Paszek M.J. (2019). Physical Principles of Membrane Shape Regulation by the Glycocalyx. Cell, 177(7), 1757-1770.e21.
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8

Lipid-based Biomaterial Preparation

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1,2-Dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt; DMPS) was purchased from Avanti Polar Lipids (Alabama, USA). Sodium phosphate monobasic (NaH2PO4, BioPerformance Certified, ≥99.0%), sodium phosphate dibasic (Na2HPO4, ReagentPlus, ≥99.0%), sodium azide (NaN3, ReagentPlus, ≥99.5%) and carbon black nanopowder were purchased from Sigma Aldrich (Poole, UK). Thioflavin T UltraPure Grade (ThT, ≥95%) was purchased from Eurogentec (Southampton, UK) and polydimethylsiloxane (PDMS) (Sylgard 184 kit) from Dow Corning (Midland, Michigan). Alexa Fluor 568 NHS-ester and Alexa Fluor 647 maleimide were purchased from Life Technologies (Carlsbad, California).
Brown J.W., Buell A.K., Michaels T.C., Meisl G., Carozza J., Flagmeier P., Vendruscolo M., Knowles T.P., Dobson C.M, & Galvagnion C. (2016). β-Synuclein suppresses both the initiation and amplification steps of α-synuclein aggregation via competitive binding to surfaces. Scientific Reports, 6, 36010.
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9

Antibody Labeling with Fluorescent Dyes

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Primary and secondary antibodies, (AF648 from R&D Systems, H00000999-M01 from Novus Biologicals, ab19348, ab7120 and ab7056 from Abcam) were labelled with amine-reactive dyes (Alexa Fluor 568 NHS ester and Alexa Fluor 647 NHS ester from Life Technologies and NHS-Fluorescein from Thermo Fisher Scientific) or TbL4 (Lumi4®-Tb-NHS, Lumiphore), both in concentration excess to the antibody solutions, in 100 mM carbonate buffer at pH 9.0. The mixtures were incubated while rotating at 25 rpm (Intelli-Mixer, ELMI) for 5 h at room temperature. The samples were purified using 30 kDa filter centrifugal devices (Amicon Ultra 0.5 mL filters) and stored in 100 mM Tris-HCl buffer, pH 7.2.
Lindén S., Singh M.K., Wegner K.D., Regairaz M., Dautry F., Treussart F, & Hildebrandt N. (2015). Terbium-based time-gated Förster resonance energy transfer imaging for evaluating protein-protein interactions on cell membranes. Dalton transactions (Cambridge, England : 2003), 44(11).
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10

Fluorescent Labeling of Oligomeric Proteins

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tau oligomer (tauO) and p53O were produced and characterized following established and published protocols [36 (link)] with an exception that recombinant p53 was heated as previously described [34 (link)]. tauO and p53 oligomer (p53O) labeling was conducted as follows: 1 mg of Alexa Fluor™ 568 NHS Ester (Invitrogen, #A20003) was dissolved in 0.1 M sodium bicarbonate to make the final concentration 1 mg/ml. The Alexa-Fluor dye was then incubated with tauO and p53O in a 1:2 (w/w) ratio. The mixture was rotated overnight at 4 °C on an orbital shaker. The following day, the solution was centrifuged (30 min, 15,000 g) using 10 kDa Amicon Ultra-0.5 Centrifugal Filter Units to remove unbound dye. The oligomers were then washed with 1× PBS until the flow-through solution was clear. The filter compartment was then flipped and centrifuged to collect the concentrate. The oligomers were reconstituted to their original volume. Alexa-Fluor labeled tauO and p53O were re-suspended in complete DMEM to obtain 0.5 or 1 μM final concentration solutions. The cells were treated with tauO or p53O for 1 or 4 h timepoints at a controlled temperature of 37 °C and 5% CO2. Afterwards, the medium was removed, and the cells collected for immunofluorescence assays.
Farmer K.M., Ghag G., Puangmalai N., Montalbano M., Bhatt N, & Kayed R. (2020). P53 aggregation, interactions with tau, and impaired DNA damage response in Alzheimer’s disease. Acta Neuropathologica Communications, 8, 132.
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