Sodium caprylate was obtained from EMD Millipore (Darmstadt, Germany). A 20% (mass/volume) stock solution was prepared by dissolving sodium caprylate in 10 mM sodium phosphate pH 6.5 solution. Sodium chloride, sodium phosphate monobasic monohydrate, sodium phosphate anhydrous, sodium phosphate dibasic heptahydrate, sodium sulfate, Tris base, Tris hydrochloride, and glycine were obtained from Avantor Performance Materials (Center Valley, PA).
Glycine
Manufactured by Avantor
Sourced in United States, China
Glycine is a crystalline, white, water-soluble amino acid commonly used in laboratory applications. It serves as a building block for various biomolecules and is a fundamental component in many biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Sodium dodecyl sulfate (sds), by Avantor (9 mentions)
Urea, by Merck Group (6 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Acrylamide, by Avantor (5 mentions)
Bromophenol blue, by Bio-Rad (5 mentions)
Sucrose, by Avantor (4 mentions)
Tween 20, by Merck Group (4 mentions)
Methanol, by Avantor (4 mentions)
Ammonium persulfate, by Merck Group (4 mentions)
Tris buffer, by Avantor (4 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Avantor (3 mentions)
Acetonitrile, by Merck Group (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Avantor (3 mentions)
Tris base, by Avantor (3 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (3 mentions)
Sodium phosphate dibasic, by Avantor (3 mentions)
Yeast extract, by BD (3 mentions)
Acrylamide, by Merck Group (3 mentions)
Tryptone, by BD (3 mentions)
Bis acrylamide, by Merck Group (3 mentions)
46 protocols using glycine
1
Preparation of Sodium Caprylate Solution
2
Lyophilized IgG1 mAb Protein Formulation
3
Quantification of Thiol Groups in Samples
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Thiol groups were measured according to the modified Ellman method. Briefly, 1.0 ml of 40 mM Ellman’s reagent (5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) solution (Serva, Heidelberg, Germany) was added to the sample (86 mM Tris (Sigma Aldrich, Saint Louis, MO, USA), 90 mM glycine (Avantor, Gliwice, Poland), 4 mM ethylenediaminetetraacetic acid (EDTA) (Avantor, Gliwice, Poland), 8 M urea (Sigma Aldrich, Saint Louis, MO, USA), 0.5% sodium dodecyl sulfate (SDS, Serva, Heidelberg, Germany), 0.2 M Tris-HCl (Sigma Aldrich, Saint Louis, MO, USA)) with pH 8.0. Next, 200 μl of each sample was added to 1.0 ml of DTNB. The samples were incubated at room temperature for 30 min. Cysteine was used (Avantor, Gliwice, Poland) as a standard, and absorbance was measured by a Perkin Elmer spectrophotometer Lambda 25 at a wavelength of λ= 412 nm (Biocompare, Baltimore, MD, USA). The concentration of -SH groups was measured from a calibration curve based on cysteine solution in PBS. The concentration of -SH groups was expressed in micromoles per milligram of whole protein in the ileal supernatant.
4
Recombinant Protein Expression and Purification
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Sodium chloride, glycerol, sodium phosphate saline (PBS), isopropyl β-d-1-thiogalactopyranoside (IPTG), Tris HCL and base, Glycine, lysozyme, dithiothreitol (DTT), and ethylene-di-aminetetraacetic acid (EDTA), Coomassie G-250 were purchased from VWR life science. Inclusion body (IB) solubilizing and protein refolding reagents: GSH, GSSG, L-Arginine, and urea were from Sigma-Aldrich (St. Louis, MI, USA)). PCR cloning kit: PET series vectors; all restriction enzymes; T4 DNA ligase and rapid protein assay BCA kit were from New England Biolabs (NEB, Ipswich, MA, USA) and Thermo Scientific (Cincinnati, OH, USA). ELISA reagents: Human serum albumin (HAS), bovine serum albumin (BSA), Tween-20, Citric acid, H2O2, o-phenylenediamine dihydrochloride (OPD) were from Sigma-Aldrich and Alfa Aesar. Protein purification apparatus and affinity chromatography columns were purchased from GE Healthcare.
5
Membrane Protein Purification and Analysis
6
Chromatin Immunoprecipitation of Neuronal Nuclei
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Extracted nuclei were cross-linked in 1% formaldehyde (AppliChem) for 5 min and quenched with 125 mM glycine (VWR). Approximately 750,000 nuclei per sample were resuspended in 500 µL PBS-T. Nuclei were stained with 1:50 Alexa Fluor488 conjugated anti-NeuN (Millipore, MAB377X) for 30 min, then washed in PBS-T. Nuclei were stored in 200 µL PBS-T, passed through a 26-G needle, and incubated with Hoechst (1:1,000) before sorting on the FACSAriaIII (BD Bioscience). Sorted NeuN+ nuclei were pelleted (4 °C, 1,250 × g, 5 min) and lysed in 750 µL RIPA buffer for 10 min. Samples were sonicated on an E220 Focused-ultra-sonicator (Covaris) for 20 min (peak power = 140 W, duty = 5, cycle/burst = 200). Low-input ChIP was carried out using the Low Cell ChIP-Seq Kit (Active Motif) using H3K27ac (Abcam, ab4729). The Next Gen DNA Library and Indexing Kit (Active Motif) were used to prepare libraries (for details, see SI Appendix ). Libraries were resuspended in 25 µL Low EDTA TE buffer then analyzed with the fragment analyzer (NGS High Sensitivity kit, Agilent) and sequenced, paired-end, on the Illumina NextSEq. 500.
7
Bacterial Hydrogen Cyanide Production Assay
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Bacteria were tested for hydrogen cyanide (HCN) production by adapting the method of Lorck (1948) [42 (link)]. Briefly, the TSA medium was amended with 0.44% glycine (VWR Chemicals, China) and 100 μL (OD600nm = 0.6) of the bacterial culture was flooded on poured agar plates with a sterile swab. A Whatman filter paper was soaked in the picric acid (0.5%) in 2% sodium carbonate for 1 min and placed below the plates’ lids. The plates were closed with parafilm and incubated at 30 °C for 96 h. The color shift of Whatman paper from yellow, conferred by sodium picrate solution, to orange or brown indicates a positive result for HCN production. The experiment was performed in triplicate.
8
Fabrication of Gelatin-Heparin Hydrogels
9
Western Blot Reagent Preparation
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Ponceau S, Ammonium Persulfate and TEMED were products of Millipore-Sigma Canada. Tween20, Tris-base, Glycine, EDTA, Methanol, SDS, Acrylamide and Bis-Acrylamide were obtained from VWR, Canada. All chemicals were of ACS grade or higher.
10
Tricolor Glutamatergic Synapse Visualization
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For the triple staining of glutamatergic synapses on PNN positive neurons, coverslips were washed three times with washing solution containing 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich; Cat. No.: F7524), 0.1 mM glycine (VWR International GmbH; Cat. No.: 101196X), and 0.1% (v/v) Triton X-100 (AppliChem; Cat. No.: A4975,0500) in PBS, after fixation. Then, following antibodies were diluted in washing solution: anti-vesicular glutamate transporter-1(vGlut) (1:1,000; guinea pig; polyclonal; Synaptic Systems GmbH; RRID: AB_887878 ), anti-postsynaptic density-95 (PSD-95) (1:500; mouse; monoclonal IgG2a; Merck KGaA; RRID: AB_1121285 ), and anti-aggrecan (1:500; rabbit; polyclonal; Merck KGaA; RRID: 90460 ). Coverslips were incubated for 1 h at room temperature. Then, coverslips were washed three times with washing solution and incubated with the secondary antibody solution for 1 h at room temperature. Here, following antibodies were used: anti-guinea pig (1:500; AF647; IgG (H+L); Jackson Immuno Research, RRID: AB_2337446 ), anti-mouse, (1:250; AF488; IgM+IgG (H+L); Jackson Immuno Research; RRID: AB_2338844 ), and anti-rabbit (1:500; Cy3; IgG (H+L); Jackson Immuno Research; RRID: AB_2338003 ). Last, coverslips were washed three times with PBS (1x), once with MilliQ water and finally mounted on microscope slides.
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