Hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O; 99%), cetyltrimethylammonium bromide (CTAB), sodium borohydride (NaBH4; 99%), silver nitrate (AgNO3; 99%), L-ascorbic acid (AA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC), sodium oleate (NaOL; >97%), hydrochloric acid (HCl, 37%), (3-mercaptopropyl)trimethoxysilane (MPTMS), ethylenediaminetetraacetic acid (EDTA), poly(ethylene glycol) methyl ether thiol (PEG-SH, Mw ≈ 5000), goat anti-human IgG, dithiotreitol (DTT), thiol-poly(ethylene glycol)amine (SH-PEG-NH2), and human serum IgG were provided by Sigma–Aldrich (St. Louis, MO). Traut’s reagent, ZebaTM spin desalting columns (7K MWCO), and Ellman’s reagent were purchased from Thermo Scientific (Rockford, IL). PEG6-NHNH2 was obtained from SensoPath Technologies (Bozeman, MT). Glass slides (7 mm × 50 mm × 0.7 mm) were from Delta Technologies (Loveland, CO).
Ellman s reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Ellman's reagent is a chemical compound used in biochemistry and molecular biology laboratories for the quantitative determination of sulfhydryl groups (–SH) in proteins and other biomolecules. It is a colorimetric assay that measures the absorbance of the resulting reaction product, providing a reliable method for analyzing the presence and concentration of thiol-containing compounds.
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Lab products found in correlation
Traut s reagent, by Thermo Fisher Scientific (3 mentions)
L cysteine, by Merck Group (3 mentions)
L ascorbic acid, by Merck Group (2 mentions)
Human serum igg, by Merck Group (2 mentions)
8 arm 20 kda peg vinyl sulfone peg vs macromers, by JenKem Technology (2 mentions)
L cysteine standard, by Thermo Fisher Scientific (2 mentions)
Ultrapure water, by Thermo Fisher Scientific (2 mentions)
Poly ethylene glycol methyl ether thiol peg sh, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Silver nitrate, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (1 mentions)
Zeba spin 7k mwco desalting columns, by Thermo Fisher Scientific (1 mentions)
Dithiotreitol dtt, by Merck Group (1 mentions)
Thiol poly ethylene glycol amine sh peg nh2, by Merck Group (1 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
L cysteine hydrochloride, by Thermo Fisher Scientific (1 mentions)
Ivis spectrum optical imaging system, by PerkinElmer (1 mentions)
4 arm peg maleimide, by JenKem Technology (1 mentions)
Poloxamer 407, by Merck Group (1 mentions)
Celltrace far red dye, by Thermo Fisher Scientific (1 mentions)
31 protocols using ellman s reagent
1
Synthesis and Functionalization of Gold Nanoparticles
2
Peptide-Functionalized PEG Hydrogels for Tissue Engineering
3
Cationized Antibody-mediated miRNA Delivery
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The cationized antibodies were obtained according to Ma’s report [17 (link)]. Briefly, the anti-CD63 antibody (Cosmobio, Tokyo, Japan) was thiolated using Traut’s reagent (Thermo Scientific, Waltham, MA, USA) in phosphate buffer (pH 8.0) containing 2 mM EDTA and then conjugated with Cys(Npys)-(D-Arg)9 (AnaSpec, Fremont, CA, USA). The anti-CD63 IgG-9r was purified by size-exclusion chromatography (Figure S2 ) and concentrated using Amicon-Ultra 40K (Merck Millipore, Burlington, MA, USA). The stoichiometry of thiol modification on the antibody was calculated based on the signals resulting from reaction with Ellman’s reagent (Thermo Scientific) (Figure S3 ). The introduction number of arginine moieties to IgG was estimated to be 2.8 molecules.
The anti-CD63 IgG-9r/anti-miR complex was obtained by mixing anti-CD63 IgG-9r and anti-miR in PBS at the indicated molar ratios and incubating the mixtures at room temperature for 20 min. The sequence of anti-miR21 [18 (link)] was as follows: 5′- UmC A A mC A U mC A G T C U G A U A A G mC U A -3′. The control sequence [18 (link)] was as follows: 5′- U T C U mC C G A A C G U G T C A mC G U T A U -3′ (plain: 2′-O-methly RNA, underlined: locked-nucleic acid (LNA)). The chemical modification patterns of those oligonucleotides are different from those of antagomirs [10 (link)].
The anti-CD63 IgG-9r/anti-miR complex was obtained by mixing anti-CD63 IgG-9r and anti-miR in PBS at the indicated molar ratios and incubating the mixtures at room temperature for 20 min. The sequence of anti-miR21 [18 (link)] was as follows: 5′- U
4
Quantification of Free Sulfhydryl Groups
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The free sulfhydryl group content of the samples was analyzed following the method of Beveridge et al. [31 (link)] and manufacturer’s instructions with some modifications. 5,5′-Dithio-bis-(2-Nitrobenzoic Acid) (DTNB) and Ellman’s reagent (Thermo Fisher Scientific, Waltham, MA, USA) were utilized to determine the content of the free sulfhydryl group in the samples. Sodium phosphate buffer (0.1 M, pH 8.0) was used to dilute the protein solution to a certain concentration. l -Cysteine hydrochloride (Alfa Aesar, Tewksbury, MA, USA) was used as a standard. Dilutions of cysteine (0.25–1.5 mM) sodium phosphate buffer (0.1 M, pH 8.0) were prepared to plot a standard curve. An amount of 50 μL of Ellman’s reagent solution (4 mg/mL) was added in the mixture of 250 μL of protein sample and 2.5 mL of sodium phosphate buffer. After incubation at room temperature for 15 min, the absorbance was measured in triplicates at 412 nm using a multimode microplate reader (Cytation 5, BioTek Instruments, Inc., Winooski, VT, USA). The free sulfhydryl group content was expressed as μmol/g protein.
5
Ellman's Reagent Glutathione Assay
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GSH measurement was performed according to published protocol [44 (link)] using Ellmans’ reagent (Thermo-Fisher Scientific).
6
Quantifying Cell Surface Thiols in Mice
7
Multimodal Hydrogel Characterization
8
Analytical Characterization of Purified Fusion Proteins
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Analytical size exclusion high-performance liquid chromatography (SE-HPLC) of purified fusion proteins was performed as described previously (19 (link), 20 (link)). Briefly, 5 μl of protein was loaded on an Agilent advance Bio SEC 300 Å, 2.7 μm, 4.6 × 300 mm SEC column (Agilent Technologies, GB) and eluted with a 0.1 ml/min flow of elution buffer (phosphate buffer) at room temperature. Protein standards (Sigma Aldrich) were also run using the same conditions mentioned above for sizing of the purified recombinant proteins. The amount of free cysteine residues was measured using Ellman's Reagent (Thermo Fisher Scientific, USA) following the manufacturer's instructions. A standard curve was constructed using known concentrations of free cysteine (Sigma-Aldrich, USA). Folding was determined in the mAb45.1 sandwich ELISA as described (19 (link), 26 (link)).
9
Cysteine Quantification and Oxygen Equilibrium of Recombinant Hemoglobin
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Ellman's reagent (5,5′-dithio-bis-(2-nitrobenzoic acid), Thermo Scientific) was used to quantify the cysteine content in the Hb samples. The reaction buffer (100 mM NaP buffer pH 8.0 + 1 mM EDTA), Ellman's reagent (71.4 µg/ml) and Hb samples were mixed and incubated at room temperature for 15 min before measurement of absorbance at 412 nm, according the manufacturer's protocol. The sample absorbance was compared to a standard curve prepared with L -cysteine (Sigma-Aldrich). The purified proteins were also analyzed by dynamic light scattering (DLS) in a Zetasizer Nano S instrument (Malvern). The Hb samples were diluted to 0.5, 1.5 and 5.0 mg/ml in 50 mM NaP buffer pH 7.2, and measured at 20 °C. First without DTT, and then incubated with addition of 10 mM DTT for 10 min and measured again. Oxygen equilibrium curves of all recombinant proteins were recorded on a Hemox Analyzer (TCS Scientific, New Hope, PA) in 10 mM potassium phosphate buffer pH 7.4 supplemented with 100 mM NaCl at 37 °C, as described previously [24] (link).
10
Analytical Size Exclusion Chromatography of Purified Proteins
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Analytical size exclusion high-performance liquid chromatography (SE-HPLC) of purified proteins was performed using an Agilent 1100 Series HPLC System (Agilent Technologies, USA) equipped with a Agilent advance Bio SEC 300 Å, 2.7 μm, 4.6 × 300 mm SEC column, (Agilent Technologies, GB) as per the manufacturer’s instructions. Briefly, five hundred pmol of protein was loaded on the SEC column and eluted with a 0.350 ml/min flow of elution buffer (phosphate buffer) at room temperature. Standard proteins from Sigma Aldrich were also run using same the conditions mentioned above for sizing of the purified recombinant proteins. The absorbance was measured at 280 nm and chromatographic peaks were integrated by HPLC ChemStation (Agilent Technologies, CA, US). The amount of free cysteine residues was measured using Ellman’s Reagent (Thermo fisher scientific, USA) following the manufacturer’s instructions. A standard curve was constructed using known concentrations of free cysteine (Sigma-Aldrich, USA).
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