Eosin isothiocyanate

Manufactured by Merck Group

Sourced in United States, Switzerland, Italy

Eosin isothiocyanate is a fluorescent dye compound commonly used in biological and biochemical research applications. It is a derivative of the eosin dye, which provides a bright pink-red color. The isothiocyanate functional group allows for covalent binding of the dye to various biomolecules, enabling their detection and visualization.

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4 protocols using eosin isothiocyanate

Synthesis and Characterization of HPW-RX40

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HPW-RX40 (2-Methoxy-4-[(E)-2-nitrovinyl]phenyl 2,3-dichlorobenzoate) was synthesized according the methods described previously [17] (link). Bovine α-thrombin was purchased from Biovision, (Mountain View, CA, USA). Collagen (Type I, equine tendon) was from Helena Laboratories (Beaumont, TX, USA). Fibrinogen, U46619 (9,11-dideoxy-9,11-methanoepoxy PGF2α), 12-O-tetradecanoylphorbol-13-acetate (TPA), A23187, eosin isothiocyanate, oxidized glutathione (GSSG), and phenyl arsenoxide (PAO) were from Sigma-Aldrich (St. Louis, MO, USA). Alexa Flour 488-conjugated phalloidin, was obtained from Molecular Probes (Eugene, OR, USA). Thapsigargin and rutin were purchased from Cayman Chemical (Ann Arbor, MI, USA). Human recombinant PDI, ERp72, and Fura-2/AM were purchased from Enzo Life Sciences (Farmingdale, NY, USA); Human recombinant ERp57 and anti-P47 Ab were from Abcam (Cambridge, UK), and ERp5 was from ProSpec Protein Specialists (Rehovot, Israel). Anti-PDI Ab was from Pierce/Thermo (Rockford, IL, USA); anti-GRP78 Ab was from Genetex (Irvine, CA, USA); phospho-PKC substrate antibody and anti-CHOP Ab were from Cell Signaling Technology (Beverly, MA, USA); anti-actin was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were purchased from Sigma Chemical Co.

Kung P.H., Hsieh P.W., Lin Y.T., Lee J.H., Chen I.H, & Wu C.C. (2017). HPW-RX40 prevents human platelet activation by attenuating cell surface protein disulfide isomerases. Redox Biology, 13, 266-277.

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Fluorescent Chitosan Nanoparticle Synthesis

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Deacetylated chitosan (medium viscosity), fluorescein isothiocyanate (FITC), fluorescein (Fluo), rhodamine isothiocyanate (Rho), eosin isothiocyanate (E), 5(6)-carboxynaphthofluorescein (CNF), 5(6)-carboxy-2′,7′-dichlorofluorescein (CCF), 7-amino-4-methyl-3-coumarinylacetic acid (CNH2), 7-hydroxy-4-methylcoumarin (COH), glycerol, Tween20, and 1–4 dioxane were all purchased from Sigma-Aldrich (Buchs, SG, Switzerland). Montmorillonite Dellite^® LVF (CEC: 105 meq/100 g) was donated by Laviosa Chimica Materia (Livorno, Italy). Fluorescamine was purchased from TCI (TCI Germany GmbH, Eschborn, Germany). Phosphate-buffered saline (PBS) (10 mM) was prepared by solubilizing NaCl (0.137 M), KCl (2.7 mM), Na₂PO₄ (0.01 M) and KH₂PO₄ (1.8 mM) in DI water. The pH was adjusted by adding NaOH and HCl.

Giovannini G., Rossi R.M, & Boesel L.F. (2021). Changes in Optical Properties upon Dye–Clay Interaction: Experimental Evaluation and Applications. Nanomaterials, 11(1), 197.

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Extraction and Analysis of Pomegranate Phytochemicals

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Bidistilled water, ethanol, 85% formic acid, ethyl acetate, glacial acetic acid, dimethyl sulfoxide (DMSO), acetonitrile RS for HPLC, punicalagin (≥98%), ellagic acid (≥95%), phosphate buffered saline (PBS), dithiothreitol (DTT), oxidized glutathione (GSSG) and eosin isothiocyanate were purchased from Sigma (Milan, Italy). EDTA (0.5 M solution pH 8.0) from IBI Scientific (Dubuque, Iowa).
Pomegranate fruits of cv. Mollar (Ml) were obtained from an Italian market. Some fruits of Italian origin, belonging to two sub-varieties of cv. Dente di Cavallo (DC₁ and DC₂) with a difference of about two weeks in the maturation stage and harvest time, were provided by a local supplier (Azienda Biologica Giovomel, Avellino, Italy). A pomegranate variety from Mozia island (South Italy) was provided by the “Missione Archeologica Mozia” of “Sapienza” University of Rome (Mz).

Altieri F., Cairone F., Giamogante F., Carradori S., Locatelli M., Chichiarelli S, & Cesa S. (2019). Influence of Ellagitannins Extracted by Pomegranate Fruit on Disulfide Isomerase PDIA3 Activity. Nutrients, 11(1), 186.

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PDI Reductase Activity Assay

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Di-E-GSSG was prepared by the reaction of eosin isothiocyanate (Sigma-Aldrich) with L-glutathione oxidised (Sigma-Aldrich Lot#100K72765) as described in detail by Raturi and Mutus, 2007 (link). Four samples of PDI (4 µg) were incubated in 20 µl 100 mM potassium phosphate (pH7), made 1.5 µM with respect to calcium and magnesium chloride, with 20 µl packed PNA-agarose beads (Vector lot ZC0504) with a capacity to bind >90 µg asialofetuin to 20 µl beads. The beads were kept at 5°C over 18 hr with intermittent mixing. Following centrifugation at 14,000 g for 5 min at 4°C and a further wash with 30 µl of buffer, the combined supernatant fluids were added to the reaction mixture to give a maximum concentration 128 nM PDI. Reductase activity was monitored as above (Raturi and Mutus, 2007 (link)). Fluorescence was measured in a Biotronix Fluorometer (Electronics and Instrumentation Services for Biological Science, University of Cambridge).

Cook G.M., Sousa C., Schaeffer J., Wiles K., Jareonsettasin P., Kalyanasundaram A., Walder E., Casper C., Patel S., Chua P.W., Riboni-Verri G., Raza M., Swaddiwudhipong N., Hui A., Abdullah A., Wajed S, & Keynes R.J. (2020). Regulation of nerve growth and patterning by cell surface protein disulphide isomerase. eLife, 9, e54612.

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