Primary amine groups of unmodified and crosslinked collagen were determined using the TNBSA assay (Thermo Scientific) according to the manufacturer's instructions. The free amine groups were quantified by comparison to a standard curve of known concentrations of glycine.
Tnbsa assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TNBSA assay is a colorimetric method used to quantify primary amines, including amino acids and proteins. It involves the reaction of 2,4,6-trinitrobenzenesulfonic acid (TNBSA) with primary amines, forming a yellow-orange colored complex that can be measured spectrophotometrically.
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Lab products found in correlation
Varioskan flash multimode reader, by Thermo Fisher Scientific (1 mentions)
Model s220, by Mettler Toledo (1 mentions)
Bca protein assay, by Merck Group (1 mentions)
Cellulose dialysis bag, by Merck Group (1 mentions)
Fluorescein, by Thermo Fisher Scientific (1 mentions)
Pen strep, by Merck Group (1 mentions)
Plasmid mega kit, by Qiagen (1 mentions)
Fetal bovine serum gibcotm, by Thermo Fisher Scientific (1 mentions)
Sybrtm green, by Thermo Fisher Scientific (1 mentions)
Agarose powder, by Bio-Rad (1 mentions)
Tripolyphosphate tpp, by Merck Group (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
4 protocols using tnbsa assay
1
Quantifying Amine Groups in Collagen
2
Quantification of Gelatin Hydrogel Crosslinking
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Crosslinking efficiency was quantified using the 2,4,6-trinitrobenzene sulfonic acid (TNBSA) assay (Thermo Fisher Scientific, Dublin, Ireland) [36 (link)]. Briefly, gelatin hydrogels (~500 mg) were incubated with TNBSA at 37 °C for 2 h. The reaction was stopped by adding 10% SDS and 1 M hydrochloric acid. The mixtures were subsequently heated at 95 °C for 15 min in order to hydrolyse the gelatin hydrogel samples. The absorbance was read at 335 nm (Varioskan Flash Multimode Reader, Thermo Fisher Scientific, Dublin, Ireland) and values were normalised to the standard curve, which has been generated with a series of known glycine concentrations (0.005, 0.01, 0.02, 0.03, 0.04 and 0.05 mg/mL).
3
Protein Recovery and Amino Group Analysis
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The pH of the samples (control and CHs) was determined using a pH meter (Model S220, Mettler Toledo GmbH, Columbus, OH, USA). The protein recovery or solubility was determined by estimating protein content using bicinchoninic acid (BCA) protein assay, according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MS, USA) with serum albumin as the standard. Free amino group content was determined via a 2,4,6-trinitrobenzene sulfonic acid (TNBSA) assay according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA) using L-leucine as the standard. Both wet and dry weights were measured to calculate the production yield.
4
Plasmid-Peptide Conjugation Protocol
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Plasmid pEGFP-N1 (Clontech, Palo Alto, CA, USA) was purified using QIAGEN Plasmid Mega Kit (Qiagen GmbH, Hilden, Germany). Cysteine terminated CDX peptide was purchased from Biomatic Co. (Wilmington, USA) with a purity percentage of 99%. Bifunctional PEG (NHS-PEG-MAL, MW 2000) was purchased from Nanocs Inc. (Boston, USA). Fluorescein was purchased from Molecular Probes (Eugene, Oregon, United States). The drug Fingolimod was purchased from (AdooQ Bioscience, Centerstone Plaza, Irvine, USA). Other purchased materials were as follows: CS (Chitolytic, 17 Carlaw Ave, Toronto, ON M4M 2R7, Canada), Acetic acid (Sigma–Aldrich, St. Louis, Missouri, USA), cell culture flasks and plates (SPL, Geumgang-ro, Pocheon-si, Gyeonggi-do, Korea), tripolyphosphate (TPP) (Sigma–Aldrich, St. Louis, Missouri, USA), cellulose dialysis bag (cut off 12-14 kDa) (Sigma–Aldrich, St. Louis, Missouri, USA), agarose powder (Bio-Rad, Hercules, California, USA), SYBRTM Green (Thermo Fisher, Waltham, Massachusetts, USA), TNBSA assay (Thermo Fisher, Waltham, Massachusetts, USA), Ellman reagent (Thermo Fisher, Waltham, Massachusetts, USA), DMEM/HG cell medium (Sigma–Aldrich, St. Louis, Missouri, USA), fetal bovine serum (GibcoTM, Thermo Fisher, Waltham, Massachusetts, USA), Pen-Strep, and Trypsin-EDTA (Sigma–Aldrich, St. Louis, Missouri, USA).
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