Alexa fluor 647 maleimide

Manufactured by Thermo Fisher Scientific

Sourced in United States

Alexa Fluor 647 Maleimide is a fluorescent dye used for labeling and detection in various biological applications. It has an excitation wavelength of 650 nm and an emission wavelength of 668 nm. The maleimide functional group allows for covalent attachment of the dye to thiol-containing biomolecules, such as proteins.

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Alexa Fluor 647 (1520 citations) Alexa 647 (424 citations) Alexa Fluor 647 C2 maleimide (51 citations) Alexa Fluors (21 citations) Alexa 647 maleimide (18 citations) Alexa Fluor 647 maleimide dyes (5 citations)

16 protocols using alexa fluor 647 maleimide

Synthesis and Conjugation of Alexa Fluor 647 Tetrazine

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To synthesize Alexa Fluor 647 tetrazine, 2.8 mg PEG dithiol (MW 3500 Da) (JenKem Technology USA, Plano, TX) and 0.41 mg methyltetrazine-PEG4-maleimide (Kerafast) were each dissolved in 1 mL dichloromethane and combined in a glass vial. 0.11 μL triethylamine (SigmaAldrich) and 1 mg Alexa Fluor-647 maleimide (Thermo Fisher Scientific) were then added and the reaction was allowed to proceed overnight at 25°C protected from light. The reaction was then precipitated in 10 mL cold diethyl ether, centrifuged at 14000 x g for 5 minutes to pellet the precipitate, and the diethyl ether was decanted. The residual diethyl ether was removed under vacuum overnight. The resulting product was dissolved at 1 mg/mL in dimethylformamide and stored at −20°C.
To fluorescently tag 200 μL microgels, Alexa Fluor 647 tetrazine was diluted in 100 μL PBS to a concentration of 0.015 mM and combined with the microgels. The mixture was then incubated at 37°C for 1 hour with agitation. Microgels were washed three times by filling tube with PBS, centrifuging at 14000 x g for 5 minutes to pellet beads, and aspirating liquid.

Truong N.F., Kurt E., Tahmizyan N., Lesher-Pérez S.C., Chen M., Darling N.J., Xi W, & Segura T. (2019). Microporous annealed particle hydrogel stiffness, void space size, and adhesion properties impact cell proliferation, cell spreading, and gene transfer. Acta biomaterialia, 94, 160-172.

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Fluorescent Labeling of Ykt6 Proteins

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The 25 μM double cysteine mutants of yYkt6 or rYkt6 proteins were incubated with 100 μM donor (Alexa Fluor 555 Maleimide, Thermo Fisher Scientific Inc), and 200 μM acceptor (Alexa Fluor 647 Maleimide, Thermo Fisher Scientific Inc) in the presence of 1 mM Tris (2-carboxyethyl) phosphine hydrochloride at 4 °C overnight. Afterward, the unreacted dyes were removed from the protein solution by SEC using a normal buffer containing 1 mM β-mercaptoethanol.

Ji J., Yu Y., Wu S., Wang D., Weng J, & Wang W. (2023). Different conformational dynamics of SNARE protein Ykt6 among yeast and mammals. The Journal of Biological Chemistry, 299(8), 104968.

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SpyCatcher003 S49C Fluorophore Labeling

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SpyCatcher003 S49C was dialyzed into TBS pH 7.2 + 1 mM TCEP to
maintain the protein in the reduced state. SpyCatcher003 S49C was
diluted to a final concentration of 100 μM in fresh TBS pH 7.2
+ 2 mM TCEP and incubated at 25 °C for 30 min. DyLight 680-maleimide
(Thermo Fisher) or Alexa Fluor 647-maleimide (Thermo Fisher) was dissolved
in anhydrous DMSO to a final concentration of 10 mg/mL, and samples
were aliquoted and stored at −80 °C until use. Dye maleimide
constructs were added to the protein at a 3-fold molar excess, with
samples rapidly pipetted to mix thoroughly, followed by rotation end-over-end
at 25 °C for 4 h, with tubes wrapped in foil to minimize light
exposure. The excess unreacted dye was quenched by addition of 1 mM
DTT and incubated at 25 °C for 1 h. Samples were centrifuged
at 16,000g for 5 min at 4 °C to remove any aggregates.
A volume of pre-swollen Sephadex G-25 resin (Sigma-Aldrich) 5-fold
greater than the volume of the labeling reaction was added to a Bio-Rad
Poly-Prep column and washed with 4 mL of PBS pH 7.4 to remove residual
storage ethanol. After the PBS pH 7.4 drained from the column, dye-labeled
samples were added to the top of the column to remove unconjugated
dye. 1 mL of PBS pH 7.4 was added to the top of the column, and 300
μL fractions were collected. Fractions 1 and 2 were pooled and
dialyzed thrice for at least 3 h in PBS pH 7.4 at 4 °C.

Keeble A.H., Wood D.P, & Howarth M. (2023). Design and Evolution of Enhanced Peptide–Peptide Ligation for Modular Transglutaminase Assembly. Bioconjugate Chemistry, 34(6), 1019-1036.

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Peptide Labeling and Purification Protocol

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Peptides were synthesized on solid phase by standard Fmoc chemistry. For cysteine labeling, the C4 peptide (RRYCKSTEL) was incubated with 5-iodoacetamide fluorescein (Sigma-Aldrich) or Alexa Fluor 647 maleimide (Thermo Fisher) in PBS DMF buffer (8.1 mM Na₂HPO₄ pH 6.5, 137 mM NaCl, 2.7 mM KCl, 1.8 mM KH₂PO₄, 20% (v/v) DMF) for 1 hr at 20 °C using a molar fluorophore excess of 1.2. Samples were purified by reversed-phase HPLC (Jasco; PerfectSil 300 ODS C₁₈) applying a linear acetonitrile gradient from 5–80% supplemented with 0.1% (v/v) TFA. Purified peptides were snap frozen in liquid nitrogen and lyophilized (Lyovac GT2, Heraeus).

Stefan E., Hofmann S, & Tampé R. (2020). A single power stroke by ATP binding drives substrate translocation in a heterodimeric ABC transporter. eLife, 9, e55943.

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Fluorescent Labeling of Suprafibrillar Aggregates

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The cysteine point mutant αS140C was labelled with AlexaFluor 647 maleimide (Thermofisher scientific, USA) in accordance with the instructions of the manufacturer. SFAs were formed (see material and methods, αS suprafibrillar aggregates) in the presence of 1 mol% of αS140C-Al647. After the SFAs were formed, Thioflavin T (ThT) (Sigma-Aldrich, USA) was added to reach a final concentration of 5 µM. An aliquot was then pipetted into custom made microscopy chambers and imaged on a Nikon Eclipse Ti microscope in confocal laser scanning mode. The ThT and AlexaFluor 647 were excited using 402 nm (CUBE, Coherent Inc., USA). The signal from the ThT dye was collected using a 450/50 nm bandpass emission filter and from the AlexaFluor 647 using a 700/75 nm bandpass filter.

Semerdzhiev S.A., Shvadchak V.V., Subramaniam V, & Claessens M.M. (2017). Non-uniform self-assembly: On the anisotropic architecture of α-synuclein supra-fibrillar aggregates. Scientific Reports, 7, 7699.

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Site-specific TRAP1 Labeling for FRET

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Two cysteines were introduced to the cysteine-free heterodimeric hTRAP1 constructs: one at the NTD at Glu140 (E140C) and another on the MD at Lys413 (K413C). Analogous constructs for the zebrafish TRAP1 was created by introducing point mutants G151C and K428C. Purified proteins were labeled with an equal mixture of Alexa Fluor 555 and Alexa Fluor 647 maleimide (ThermoFisher, Waltham, MA) at 2X molar excess to cysteines and incubated overnight at 4˚C. Unreacted dyes were then quenched with addition of 5 mM β-MeOH and removed using HiTrap desalting column (2 × 5 mL). For FRET experiments, 0.5 µM of labeled heterodimers were used. Measurements were obtained using a Horiba Jobin Yvon FluoroMax four spectrofluorometer equipped with a chilling/heating water bath. Samples were excited at 532 nm and emission wavelengths were collected at 567 nm and 668 nm for donor and acceptor fluorescence, respectively. Relative FRET efficiencies are calculated by taking the ratio of acceptor to donor fluorescence intensity. The change in FRET is the change of this ratio relative to timepoint 0.

Elnatan D., Betegon M., Liu Y., Ramelot T., Kennedy M.A, & Agard D.A. (2017). Symmetry broken and rebroken during the ATP hydrolysis cycle of the mitochondrial Hsp90 TRAP1. eLife, 6, e25235.

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Alexa Fluor 647 Labeling of Cys-DogCatcher

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Dye labeling took place with tubes wrapped in foil, to minimize light exposure. Alexa Fluor 647-maleimide (Thermo Fisher) was dissolved in DMSO to 10 mg/mL. Cys-DogCatcher was dialyzed into TBS pH 7.4 and reduced for 30 min at 25°C with 1 mM TCEP [tris(2-carboxyethyl)phosphine)]. 100 μM Cys-DogCatcher was incubated with a 3-fold molar excess of dye:protein and reacted with end-over-end rotation at 25°C for 4 hr. After quenching the unreacted maleimide with 1 mM DTT for 30 min at 25°C, samples were centrifuged at 16,000 g for 5 min at 4°C to remove any aggregates. Free dye was removed using Sephadex G-25 resin (Merck) and dialyzing thrice each time for at least 3 hr in PBS pH 7.4 at 4°C.

Keeble A.H., Yadav V.K., Ferla M.P., Bauer C.C., Chuntharpursat-Bon E., Huang J., Bon R.S, & Howarth M. (2022). DogCatcher allows loop-friendly protein-protein ligation. Cell Chemical Biology, 29(2), 339-350.e10.

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Selective Binding of Cry3Aa Crystals to Helicobacter pylori

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Purified Cry3Aa and Cry3Aa‐PX (X = 7, 8, and 17) fusion crystals were labeled with Alexa Fluor™ 647 maleimide (ThermoFisher). H. pylori and E. coli were stained with Hoechst 33342. The labeled crystals (1 µm) was incubated with either Hoechst 33342‐stained bacteria seeded at 10⁸ CFU in a 12‐well plate, or GES‐1 cells seeded at 10⁵ cells per well in a 6‐well plate for 4 h at 37 °C. At the end of the incubation period, the treated GES‐1 cells were washed with 2 U mL⁻¹ heparin in PBS, trypsinized, and washed extensively with PBS while the treated bacteria were fixed in 4% paraformaldehyde followed by extensive washing. Cells were then analyzed on a BD FACSVerse flow cytometer. The binding specificity of each crystal construct was determined based on the percentage of cells with bound labeled crystals.
The targeting selectivity, defined as the selectivity of Cry3Aa or Cry3Aa‐PX crystals for H. pylori over GES‐1 cells or E. coli was determined by flow cytometric analysis and calculated based on the percent of crystal‐bound H. pylori relative to that of the other organism.
For GES‐1 cells, the H. pylori targeting selectivity was adjusted for the difference in their surface areas. The formula used was:

\begin{matrix} H . pylori targeting selectivity = (% of H . pylo ri binding with crystals / \\ % of GES - 1 binding with crystals) (surface area of GES - 1 / \\ surface area of H . pylori) \end{matrix}

Zhang W., Yang Z., Zheng J., Fu K., Wong J.H., Ni Y., Ng T.B., Cho C.H., Chan M.K, & Lee M.M. (2023). A Bioresponsive Genetically Encoded Antimicrobial Crystal for the Oral Treatment of Helicobacter Pylori Infection. Advanced Science, 10(30), 2301724.

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Synthesis and Characterization of Functional Polymers

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Butyl methacrylate, 4-cyano-4-(phenocarbonothioylthio)pentanoate, poly(ethylene glycol) methyl ether (M_n = 2,000 Da), dicyclohexylcarbodiimide, 4-dimethylaminopyridine, and dichloromethane were purchased from Sigma-Aldrich. 2-(diethylaminoethyl) methacrylate was purchased from TCI Chemicals. 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) was purchased from Wako Chemicals. Pyridyl disulfide ethyl methacrylate was synthesized as described previously (36, 40 (link)). 2′3′-cGAMP was synthesized as described previously (36 (link)). Alexa Fluor 647 Maleimide was purchased from Thermo Fisher Scientific.

Florian D.C., Bennett N.E., Odziomek M., Baljon J.J., Wehbe M., Merkel A.R., Fischer M.A., Savona M.R., Rhoades J.A., Guelcher S.A, & Wilson J.T. (2023). Nanoparticle STING Agonist Reprograms the Bone Marrow to an Antitumor Phenotype and Protects Against Bone Destruction. Cancer Research Communications, 3(2), 223-234.

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Redox Immunohistochemistry for Thiol Labeling

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Assays were performed as previously described, according to a procedure we refer to as redox immunohistochemistry [26 (link),27 (link)]. Briefly, equal amounts of cells were harvested and suspended in a lysis solution containing Tris-HCl 50 mM, SDS 1%, EDTA 1 mM, NEM 10 mM (N-Ethylmaleimide, Sigma-Aldrich, St. Louis, Mo, USA, E3876), Alexa Fluor 647 Maleimide 20 uM (Thermo Scientific™, Waltham, MA, USA, A20347), and proteinase inhibitors mix to label reduced thiols. Lysates were incubated for 5 min at 70 °C, sonicated, and finally incubated for 30 min at RT in the dark. Quenching of unreacted thiols was performed with 100 mM NEM for 30 min. Protein extracts were then precipitated with a cold precipitation solution (50% acetone, 25% methanol, and 25% ethanol). Next, disulfide bonds were reduced with 20 mM TCEP (Tris(2-carboxyethyl)phosphine, Thermo Scientific™, Waltham, MA, USA, 20490). Newly reduced thiols were labelled with AlexaFluor 555 Maleimide 20 uM (Thermo Scientific™, Waltham, MA, USA, A20346). After precipitation, samples were diluted in Laemmli sample buffer, and proteins were resolved under reducing conditions. After electrophoresis, the gel was fixed with 5orthophosphoric 2.5% orthophosphoric acid overnight under mild agitation. Final images and analysis were performed with the Chemidoc MP Imaging System (Bio-Rad Laboratories S.r.l., Hercules, CA, USA).

Altintas D.M., Gallo S., Basilico C., Cerqua M., Bocedi A., Vitacolonna A., Botti O., Casanova E., Rancati I., Milanese C., Notari S., Gambardella G., Ricci G., Mastroberardino P.G., Boccaccio C., Crepaldi T, & Comoglio P.M. (2022). The PSI Domain of the MET Oncogene Encodes a Functional Disulfide Isomerase Essential for the Maturation of the Receptor Precursor. International Journal of Molecular Sciences, 23(20), 12427.

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