Wheat gliadin (Sigma-Aldrich) was prepared in 100 mM glycine pH 2.5 and variable concentrations of pepsin (0.05–10 μM) from porcine gastric mucosa (Fluka), neprosin (0.05–2 μM), or mixtures of 0.5 μM pepsin and 0.05–2 μM neprosin were used to digest 10 mg/mL gliadin slurries. Reactions were monitored by turbidimetry in 96-well plates (Corning) at 37 °C in a microplate spectrophotometer (BioTek). Reactions were quenched by boiling in SDS sample buffer before analysis by SDS-PAGE. Gliadin degradation by neprosin was also analysed by zymography using SDS-PAGE gels containing either Wheat gliadin or teleostean gelatin (Sigma-Aldrich), which was used as a control, at 0.1 mg/mL. Pro-neprosin was also tested, which became activated to the mature form during the assay. Proteins were renatured by washing the zymograms with 2.5% Triton X-100 in 100 mM glycine pH 2.5, 200 mM sodium chloride. After further washes with the same buffer plus 0.02% Brij-35, the zymograms were incubated overnight in the same buffer, rinsed briefly with water, and stained with Coomassie.
Wheat gliadin
Manufactured by Merck Group
Sourced in Italy
Wheat gliadin is a laboratory product used for analytical purposes. It is a protein fraction extracted from wheat that is commonly used as a reference material or standard in various laboratory analyses.
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5 protocols using wheat gliadin
1
Gliadin Digestion by Pepsin and Neprosin
2
SDS-PAGE Zymogram Analysis of Gliadin Degradation
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Gliadin degradation was assessed with 12% SDS-PAGE zymogram gels containing wheat gliadin (0.6 mg/mL; Sigma, St. Louis, MO), without β-mercaptoethanol. The protein samples were diluted (1/20) in 10 mM Tris–HCl (pH 7.5), and electrophoresis was performed at 100 V at 4 °C. Protein renaturing in the gels was achieved by washing twice for 30 min at room temperature in renaturing buffer containing 2% (v/v) Triton-X-100, 0.1 M NaCl, and 0.05 M Tris–HCl (pH 7.8). Subsequently, the Triton-X-100 was removed by washing (3 × 20 min) in developing buffer (0.05 M Tris–HCl, pH 7.8). After overnight in developing buffer at 37 °C, gels were stained for 30 min with 0.1% (w/v) Coomassie Brilliant Blue R-250.
3
Gliadin Hydrolysis and Preparation
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To obtain PTG, commercially available gliadin was digested as previously described by Drago et al. [26 (link)]. Briefly, 50 g wheat gliadin (Sigma-Aldrich, Milan, Italy) was dissolved in 500 mL 0.2 N HCl for 2 h at 37 °C with 1 g pepsin (Sigma-Aldrich, Milan, Italy). The resultant peptic digest was further digested by addition of 1 g trypsin (Sigma-Aldrich, Milan, Italy) after pH adjusted to 7.4 using NaOH 2 M. The solution was stirred vigorously at 37 °C for 4 h, and then boiled (100 °C) for 30 min, freeze-dried, lyophilized, and stored at −20 °C until used.
4
Gliadin Degradation Assay by SDS-PAGE
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Gliadin degradation was assessed as previously described [12 (link)] using 12% SDS-PAGE zymogram gels containing wheat gliadin (0.6 mg/mL; Sigma, St. Louis, MO, USA), without β-mercaptoethanol. Protein samples were diluted 1/20 in 10 mM Tris-HCl (pH 7.5) and run in electrophoresis gel at 100 V and 4 °C. Afterwards, renaturation of proteins was performed by washing the gels twice for 30 min in renaturing buffer containing 2% (v/v) Triton-X-100, 0.1 M NaCl, and 0.05 M Tris-HCl (pH 7.8). Next, gels were maintained in developing buffer at 37 °C overnight and stained for 30 min with 0.1% (w/v) Coomassie Brilliant Blue R-250 [12 (link)].
5
Gliadin Peptide Extraction Protocol
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PTG was obtained following a previously published procedure [34 (link)]. Briefly, 50 g wheat gliadin (Sigma-Aldrich, Milan, Italy) was dissolved in 500 mL 0.2 N HCl and digested for 2 h at 37 °C with 1 g pepsin (P6887, Sigma-Aldrich, Milan, Italy). After adjusting to pH 7.4, the peptic digest was incubated with 1 g trypsin (T7418, Sigma-Aldrich, Milan, Italy) for 4 h at 37 °C. After boiling for 30 min, the solution was freeze-dried, lyophilized, and stored at −20 °C until used.
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