Zymogram gels (6% or 8%) containing gliadin from wheat (Sigma, St Louis, MO) as the incorporated substrate were prepared as previously described (23 (link),24 ). Bacterial cell suspensions, diethylaminoethanol (DEAE) fraction F4, or purified pseudolysin (Elastin Products Company, Owensville, MO), isolated by a published method (25 (link)), were tested for activity. Electrophoresis was carried out at 100 V at 4 °C. Gels were renatured and developed in Novex buffers at pH 7.0 (InVitrogen, Carlsbad, CA) as described (21 (link),23 (link)). For some gels, the renaturing and developing buffers were adjusted to pH 4.0, 3.0, and 2.0, with HCl. For other gels, 5 mm EDTA was added to both buffers at pH 7.0. After 48 h of development at 37 °C, gels were stained with 0.1% Coomassie blue in 40% (v/v) methanol and 10% (v/v) acetic acid, and destained.
Gliadin from wheat
Manufactured by Merck Group
Sourced in United States
Gliadin is a protein found in wheat. It is used in laboratory settings as a reference material for the identification and analysis of gluten proteins.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Merck Group (4 mentions)
Pepsin, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
T9033, by Merck Group (1 mentions)
Pt gliadin, by Merck Group (1 mentions)
G3375, by Merck Group (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Euroclone (1 mentions)
Gliadin, by Merck Group (1 mentions)
6 hydroxycoumarin, by Merck Group (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Ethyl alcohol, by Merck Group (1 mentions)
Ovalbumin (ova), by Thermo Fisher Scientific (1 mentions)
G 50 columns, by GE Healthcare (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Alexa fluor microscale labelling kits, by Thermo Fisher Scientific (1 mentions)
Resveratrol, by Fujifilm (1 mentions)
10 protocols using gliadin from wheat
1
Gliadin Zymogram Gel Protocol
2
Intestinal Inflammation Induction Protocol
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Each intestine section was infused with serum-free tissue culture medium containing Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco, CA, USA) supplemented with 20% KnockOut serum replacement (Gibco), 2% B-27 and 1% of N-2 supplements (Gibco), 1% L-glutamine, 1% non-essential amino acids (NEAA), 1% HEPES and stimulated with Thapsigargin (0.5 or 5.0 µg/ml, T9033 Sigma-Aldrich) or PT-Gliadin (peptic tryptic digest Gliadin; 2.5 mg/ml, Sigma-Aldrich). The tissue culture medium was loaded into 5 ml syringe (for short-term experiments, 16 h) and infused into the device input ports by a syringe pump (flow rate of 99ul/h) (as described by Yissachar N. et al. with minor modification).
Gliadin from wheat (Sigma-Aldrich) was digested, as previously described15 (link). In detail, each 50 g gliadin (G-3375, Sigma-Aldrich) was firstly dissolved in 500 mL 0.2 N HCl for two hours at 37 °C with 1 g pepsin (Sigma-Aldrich). The resultant peptic digest was further digested by the addition of 1 g trypsin (Sigma-Aldrich), after pH adjusted to 7.4 using 2 N NaOH; next, the solution was incubated at 37 °C for four hours with a vigorous agitation. Finally, the mixture was boiled to inactivate enzymes for 30 min and was stored at −20 °C (hereafter referred as PT-Gliadin).
Gliadin from wheat (Sigma-Aldrich) was digested, as previously described15 (link). In detail, each 50 g gliadin (G-3375, Sigma-Aldrich) was firstly dissolved in 500 mL 0.2 N HCl for two hours at 37 °C with 1 g pepsin (Sigma-Aldrich). The resultant peptic digest was further digested by the addition of 1 g trypsin (Sigma-Aldrich), after pH adjusted to 7.4 using 2 N NaOH; next, the solution was incubated at 37 °C for four hours with a vigorous agitation. Finally, the mixture was boiled to inactivate enzymes for 30 min and was stored at −20 °C (hereafter referred as PT-Gliadin).
3
Caco-2 Cells Exposure to Digested Gliadin
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Caco-2 cells from the American Type Culture Collection (ATCC) were cultured and maintained in DMEM (Euroclone, Milano, Italy) supplemented with 10% FBS, 100 units/mL penicillin, 0.1 mg/mL streptomycin and 1% l -glutamine and kept at 37 °C in a 5% CO2 air atmosphere. gliadin from wheat (Sigma, St. Louis, MO, USA) was digested as described [40 (link)] to a final concentration of 1 g/mL. In detail, gliadin was firstly dissolved in 500 mL 0.2 N HCl for 2 h at 37 °C with 1 g of pepsin (Sigma). The resultant peptic digest was further digested by the addition of 1 g trypsin (Sigma), after pH adjustment to 7.4 using 2 N NaOH, and the solution was incubated at 37 °C for 4 h with vigorous agitation. Finally, the mixture was boiled to inactivate enzymes for 30 min and stored at −20 °C. For in vitro assays, digested gliadin (10 mg/mL) was firstly administered directly to Caco-2 cells in suspensions (about (1–1.5) × 106) for 30 min at 37 °C, and then cells were distributed in multiwell 12-wells plates for different time intervals.
4
Biomaterial Synthesis and Characterization
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Gliadin from wheat, ethyl alcohol, 6-hydroxy coumarin, and trehalose dihydrate (anhydrous) was obtained from Sigma-Aldrich (St. Louis MO, USA). Sodium hydroxide, citric acid, sodium citrate, Pluronic F-127, a BCA assay kit (Pierce, Thermo Scientific, Chicago, IL, USA), Dulbecco’s modified Eagle’s media (DMEM, GIBCO®), fetal bovine serum (Atlanta Biologicals Inc., Lawrenceville, GA, USA), and penicillin/streptomycin (GIBCO™) were from Fisher Scientific Ltd. (Loughborough, UK). FITC tagged IgG was from Molecular Probes (Eugene, OR, USA). Mouse IgG was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Green fluorescent non-functionalized polystyrene particles were from Polyscience Inc. (Washington, PA, USA). Ficoll–Paque™ was purchased from GE health care (Piscataway, NJ, USA). All the other chemicals and solvents were analytical grade from Sigma-Aldrich Co. (St. Louis, MO, USA).
5
Characterization of Wheat Gliadin Digestion by Anthocyanin
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Gliadin from wheat, pepsin, and trypsin were purchased from Sigma-Aldrich (Budapest, Hungary). Sour cherry anthocyanin extract (AC) was prepared by the Department of Feed and Food Biotechnology, University of Debrecen (Hungary), as described previously [16 (link)]. The main anthocyanin components in the extract were cyanidin-3-O-glucosyl-rutinoside, cyanidin-3-O-rutinoside, and cyanidin-3-O-monoglucoside. The anthocyanin components of the AC extract in the ‘Újfehértói fürtös’ variety were determined by UHPLC. Based on the molar ratio of the anthocyanin components, we calculated the average molar mass of AC, for which we obtained a value of 574.5 g/mol [17 (link)]. This value was used to calculate the μM concentrations in experiments. All other reagents were ordered from Sigma-Aldrich (Budapest, Hungary).
6
Peptic-Tryptic Digestion of Wheat Gliadin
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Gliadin from wheat (Sigma) was digested, as described [18 (link)]. In detail, whole gliadin (1 g/mL) was firstly dissolved in 500 mL 0.2 N HCl for two hours at 37 °C with 1 g pepsin (Sigma). The resultant peptic digest was further digested by addition of 1 g trypsin (Sigma), after pH adjusted to 7.4 using 2 N NaOH; next, the solution was incubated at 37 °C for four hours with a vigorous agitation. Finally, the mixture was boiled to inactivate enzymes for 30 min and was stored at −20 °C (hereafter referred as PT-gliadin). Ovalbumin (Sigma) at 1 g/mL was used as an additional external protein for internalization and cell toxicity studies. PT-gliadin or Ovalbumin were then administered directly to the cells. For PT-gliadin Alexa Fluor 488 or 555 labeling, purification of unlabeled dyes was performed using affinity chromatography-purification G50 columns (GE Healthcare, Little Chalfont, UK). The resulted proteins were resuspended in PBS and quantified through Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA). Then, PT-gliadin or Ovalbumin (100 µg each) proteins were labeled with Alexa Fluor 488 or Alexa Fluor 555 by specific Alexa Fluor Microscale labelling kits (ThermoFisher, Waltham, MA, USA), according to manufacturer’s instructions.
7
Extraction and Purification of ATI
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Resveratrol (98%) was bought from FUJIFILM Wako (Osaka, Japan). The p31-43 peptides (LGQQQPFPPQQPY) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Extraction and purification of ATI were prepared as described by Zevallos (18 (link)). Gliadin from wheat was purchased from Sigma-Aldrich (Sigma, Missouri, USA).
8
Evaluating Gliadin-Doxorubicin Cytotoxicity
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Gliadin from wheat, 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide salt (used for MTT test), phosphate buffered saline tablets (PBS), dimethyl sulfoxide and amphotericin B solution (250 µg/mL) were all obtained from Sigma Aldrich (Milan, Italy). DOX was purchased from DBA Italia S.r.l. (Segrate, Milan, Italy) whereas Super Refined polyoxyethylene (2) oleyl ether was obtained from Croda International (Snaith, UK). Ethanol was purchased from Carlo Erba SpA (Milan, Italy). Dulbecco’s modified Eagle’s culture medium (DMEM) enriched with glutamax I, trypsin/ethylene diamine tetraacetic acid (EDTA), penicillin/streptomycin solution and fetal bovine serum (FBS), used during in vitro studies, were purchased from GIBCO (Life Technologies, Monza, Italy). Human breast adenocarcinoma cells (MCF-7) were obtained from the IRCCS Azienda Ospedaliera Universitaria San Martino—IST Istituto Nazionale per la Ricerca sul Cancro. Spectrum Laboratories Inc. (Eindhoven, The Netherlands) provided the cellulose acetate membrane used for in vitro release studies (cut-off 10 kDa).
9
Synthesis and Characterization of Gliadin-based Polymers
10
Purification and Characterization of Wheat Allergen ATI
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The extraction and purification of ATI were prepared as described by Zevallos, V. F [20 (link)]. Briefly, high-gluten wheat flour (Triticum aestivum L.) was extracted by using ammonium bicarbonate buffer, the supernatant was fractionally precipitated by using ammonium sulfate, and then the mixture was dialyzed, sterile filtrated, lyophilized and further purified by FPLC. Gliadin from wheat, bovine serum albumin (BSA), Poly:IC and DSS (MW: 40,000 Da) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Sodium salt of Caboxy Methyl Cellulose (CMC) with food grade was obtained from Jiangsu Tailida New Materials CO., Ltd. (Jiangsu Tailida, Nantong, China). Cholera Toxin (CT, B subunit) and tissue lysate were acquired from Absin (Absin, Shanghai, China). Mouse IL-1β, TNF-α, IL-4 and IL-13 enzyme-linked immunosorbent assay kits were purchased from Luminex (Univ-bio, Shanghai, China). APC anti-mouse IL-4, PE/Cyanine 7 anti-mouse CD4, FITC anti-mouse INF-γ and Fixable Viability Stain 510 were procured from BD Pharmingen (BD Biosciences, Piscataway, NJ, USA). Other analytical grade reagents were purchased from Xilong Scientific (Xilong Scientific, Guangdong, China).
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