2 4 6 trinitrobenzenesulfonic acid tnbs

Manufactured by Thermo Fisher Scientific

Sourced in Ireland, United States

2,4,6-Trinitrobenzenesulfonic acid (TNBS) is a chemical compound used in various laboratory applications. It serves as a reagent for the determination of primary amino groups in proteins and other biomolecules. TNBS reacts with primary amines to produce colored derivatives, allowing for spectrophotometric quantification of amino group content.

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5 protocols using 2 4 6 trinitrobenzenesulfonic acid tnbs

Enzymatic Hydrolysis of Whey Protein

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Whey protein concentrate containing ~80% (w/w) protein (WPC80) was kindly provided by Carbery (Balineen, Cork, Ireland). The food-grade enzyme preparations used were Alcalase^® 2.4 L (2.4 Anson units/g) and Prolyve1000^® (≥3000 U/g protein), obtained from Sigma-Aldrich (Dublin, Ireland) and Soufflet Biotechnologies (Caen, France), respectively. Trifluoroacetic acid (TFA), Trolox and 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) were purchased from Sigma-Aldrich. High-pressure liquid chromatography- (HPLC) and mass spectrometry (MS)-grade water and acetonitrile (ACN) were provided by Fisher Scientific (Dublin, Ireland). 2,4,6-Trinitrobenzenesulfonic acid (TNBS) was obtained from Pierce Biotechnology (Medical Supply, Dublin, Ireland). Food-grade sodium hydroxide (NaOH) was obtained from Micro-Bio Ireland Ltd. (Cork, Ireland).

Kleekayai T., O’Neill A., Clarke S., Holmes N., O’Sullivan B, & FitzGerald R.J. (2022). Contribution of Hydrolysis and Drying Conditions to Whey Protein Hydrolysate Characteristics and In Vitro Antioxidative Properties. Antioxidants, 11(2), 399.

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Enzymatic Hydrolysis of Milk Proteins

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The milk protein substrates NaCN (Arrabawn Co-Operative Creamery, Nenagh, Ireland), MPC (Kerry Ingredients, Listowel, Ireland) and WPC (Carbery, Ballineen, Ireland) had protein contents of 85%, 84% and 82% (w/w), respectively, and were kindly provided by the above manufacturers. The food-grade proteolytic preparation Debitrase HYW20™ was kindly provided by Rhodia Ltd. (Cheshire, UK), and Prolyve 1000™ was provided by Lyven Enzymes Industrielles (Caen, France). 2,4,6-Trinitrobenzenesulfonic acid (TNBS) was obtained from Pierce Biotechnology (Medical Supply, Dublin, Ireland) and all other chemicals and reagents were purchased from Sigma Chemical Company Ltd. (Dublin, Ireland).

Gruppi A., Dermiki M., Spigno G, & FitzGerald R.J. (2022). Impact of Enzymatic Hydrolysis and Heat Inactivation on the Physicochemical Properties of Milk Protein Hydrolysates. Foods, 11(4), 516.

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Whey Protein Enzymatic Hydrolysis

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Whey protein concentrate (WPC80, 80.98% ± 0.68% (w/w) protein (determined by the Kjeldahl nitrogen determination method)) was obtained from Carbery Group (Balineen, Cork, Ireland). FlavorPro^®Whey 750P (>55 casein protease U/g) was obtained from Biocatalysts Ltd. (Cefn, Wales, UK) and Debitrase^® HYW20 (11,470 U/g) was obtained from DuPont-Danisco (Marlborough, Wiltshire, UK). ABTS, trifluoroacetic acid (TFA), Trolox, 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH), mass spectrometry (MS) grade water and acetonitrile were purchased from Sigma-Aldrich (Dublin, Ireland). Sodium hydroxide (NaOH) and high pressure liquid chromatography (HPLC) grade water and acetonitrile were provided by Fisher Scientific (Dublin, Ireland). 2,4,6-Trinitrobenzenesulfonic acid (TNBS) was obtained from Pierce Biotechnology (Medical Supply, Dublin, Ireland). Dulbecco’s minimum essential medium (DMEM), fetal bovine serum (FBS), antibiotic-antimycotic solution, L-glutamine, Dulbecco’s phosphate buffered saline (PBS), Hank’s balanced salt solution (HBSS), 2’,7’-dichlorofluorescein-diacetate (DCFH-DA) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Wicklow, Ireland).

Kleekayai T., Le Gouic A.V., Deracinois B., Cudennec B, & FitzGerald R.J. (2020). In Vitro Characterisation of the Antioxidative Properties of Whey Protein Hydrolysates Generated under pH- and Non pH-Controlled Conditions. Foods, 9(5), 582.

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Hyaluronic Acid Modification Protocol

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Unless otherwise noted, all chemicals and solvents were of analytical grade and used as provided by the manufacturers. Sodium periodate, hyaluronic acid (sodium salt, MW: 1500 -1800 kDa), tert-butyl carbazate (t-BC), tri-Boc hydrazinoacetic acid, HATU, 4-methylmorpholine, trifluoroacetic acid (TFA), anhydrous dimethylformamide (DMF), and dichloromethane (DCM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,4,6-trinitrobenzene sulfonic acid (TNBS) was purchased from Thermo Scientific Inc. (Odessa, TX, USA).

Zhu D., Wang H., Trinh P., Heilshorn S.C, & Yang F. (2017). Elastin-like Protein-Hyaluronic acid (ELP-HA) Hydrogels with Decoupled Mechanical and Biochemical cues for Cartilage Regeneration. Biomaterials, 127, 132-140.

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Necroptosis Pathway Protein Analysis

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Caspase-3, Caspase-8, RIPK1, RIPK3 (rabbit antibody), MLKL, OGT and OGA antibodies were purchased from Proteintech (Wuhan, China). Phospho-RIPK1, phospho-RIPK3, phospho-MLKL and secondary antibodies were from Cell Signaling Technology (Beverly, MA, USA). MPO antibody was provided by ABclonal Technology (Wuhan, China). CD68 antibody was from Abcam (Cambridge, MA). O-GlcNAc antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Another RIPK3 (mouse) antibody was purchased from Novus Biologicals (Littleton, CO, USA). 2,4,6-trinitrobenzene sulfonic acid (TNBS) was from Thermo Fisher Scientific (CA, USA). Protein A/G magnetic beads were purchased form Bimake (Shanghai, China). The TUNEL staining and immunohistochemistry kits were purchased from Wuhan Gugeshengwu Technology Co.,Ltd. (Wuhan, China). All other regular reagents were from Wuhan Gugeshengwu Technology Co.,Ltd. unless otherwise specified.

Wu F., Shao Q., Cheng Z., Xiong X., Fang K., Zhao Y., Dong R., Xu L., Lu F, & Chen G. (2021). Traditional herbal formula Wu-Mei-Wan alleviates TNBS-induced colitis in mice by inhibiting necroptosis through increasing RIPK3 O-GlcNAcylation. Chinese Medicine, 16, 78.

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