The study was performed on serum from screened donors secured from the Australian Red Cross. Three serum biological replicates were analyzed in the study. Since it is the simplest amino acid with a molecular weight of 75 Da, glycine was used as the reference molecule and its analytical grade counterpart was purchased from Sigma-Aldrich (G7126, St. Louis, MO, USA). The whole serum samples were augmented with dynamic range of concentrations of glycine; 0, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 17.5, 25, and 50 mg/mL. Thirteen sample sets were generated from each individual biological replicate resulting in 39 sample sets. A glycine solution was prepared with a concentration of 200 mg/mL in 0.9% NaCl solution (Baxter, Deerfield, IL, USA). Similarly, individual samples of serum albumin and globulins (Sigma-Aldrich, USA) were prepared in 0.9% NaCl solution (Baxter, USA). Furthermore, 100 µL of serum samples containing varying concentrations of glycine was deposited on different coverslips (24 × 55 mm with a thickness of 0.12–0.17 mm made from high-quality glass were purchased from Matsunami, Bellingham, WA, USA) for spectroscopic analysis. Briefly, the samples were dried at room temperature for 30 min and further blow-dried with a hair dryer to remove the majority of bound water. The same sample sets were used to build both ATR and NIR spectral data sets.
G7126
Manufactured by Merck Group
Sourced in United States, Italy
The G7126 is a precision laboratory instrument manufactured by Merck Group. It is designed for accurate and reliable measurements in a laboratory setting. The core function of the G7126 is to provide precise data and analysis that supports scientific research and product development.
Automatically generated - may contain errors
Lab products found in correlation
G5882, by Merck Group (3 mentions)
Pbs 1x, by Merck Group (2 mentions)
G2500, by Merck Group (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Nacl solution, by Baxter (1 mentions)
L5501, by Merck Group (1 mentions)
D9663, by Merck Group (1 mentions)
Mouse anti calbindin d 28 k, by Swant (1 mentions)
H1009, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Sodium borohydride, by Merck Group (1 mentions)
S2889, by Merck Group (1 mentions)
S6672, by Merck Group (1 mentions)
F8775, by Merck Group (1 mentions)
Sodium thiosulfate, by Merck Group (1 mentions)
Silver nitrate, by Merck Group (1 mentions)
M3262, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
Petri dishes, by Corning (1 mentions)
Fb002, by Thermo Fisher Scientific (1 mentions)
8 protocols using g7126
1
Glycine-Spiked Serum Analysis
2
Immunohistochemical Staining of Cerebellar Calbindin
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The cerebellar sections were washed three times in 0.1 M PB pH7.4 on ice for 10 min and incubated in 0.3% hydrogen peroxide (H1009, Sigma-Aldrich) in the same buffer for 30 min on ice. After three washes with the same buffer again, sections were incubated in 0.5% sodium borohydride (#71320, Sigma-Aldrich) for 30 min at room temperature, followed by three 10 min washes with 0.1 M PB. The sections were incubated in blocking buffer containing 1% BSA, 0.01% glycine (G7126, Sigma-Aldrich), 0.01% lysin (L5501, Sigma-Aldrich), 1% normal donkey serum (D9663, Sigma-Aldrich), 0.05% Triton X-100 (#22146, EMS), 0.1% cold water fish gelatin (#25560, EMS) in 0.1 M PB for 2 h on ice. Samples were stained with primary antibody, mouse anti-Calbindin-D28 K (#AgCB10 abs, Swant, Switzerland), at 1:5,000 dilution in the same blocking buffer, at 4°C overnight. The following day, slices were washed four times in 0.1 M PB on ice and probed with secondary antibody, donkey anti-mouse conjugated to Alexa Fluor 488 (A-21202, Invitrogen) at 1:200 dilution in blocking buffer for 2 h on ice.
3
Silver Staining Protocol for Protein Gels
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Gels were fixed for 45 min at room temperature in a fixation solution of 30% (v/v) ethanol (VWM Chemicals, 20821.296), 10% (v/v) acetic acid, and subsequently sensitized for 45 min at RT in a sensitizing solution of 7.5% (v/v) ethanol, 6.8% (w/v) sodium acetate (Sigma Aldrich, S2889), 0.2% (w/v) sodium thiosulfate (Sigma Aldrich, S6672), and 0.03125 % glutaraldehyde (v/v) (Sigma Aldrich, G5882). Gels were then washed three times for 5 min with Milli‐Q water at room temperature and incubated for 20 min at room temperature in a silver staining solution of 0.2% w/v silver nitrate (Sigma Aldrich, 31630) and 0.0028% formaldehyde (v/v) (Sigma Aldrich, F8775). The gels were washed for 30 s with Milli‐Q water and immersed in a developing solution of 2.5% (w/v) sodium carbonate (Sigma Aldrich, S2889) and 0.0014 % (v/v) formaldehyde for 5–7 min at room temperature. The reaction was stopped with a stopping solution of 1% (w/v) glycine (Sigma Aldrich, G7126) and washed three times for 5 min with Milli‐Q water at room temperature. All steps were performed using 50 ml of solution.
4
NMDA-Mediated Calcium Signaling in Motor Neurons
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On day 28 in vitro (MNs-DIV 0), MNPs were seeded in Matrigel (Corning #354230)-coated glass Petri dishes and cultured with HopCell® Human MN differentiation culture medium. The culture medium was subsequently replaced with the neurobasal medium. By DIV 30, we performed calcium signal recording and N-methyl-D-aspartate (NMDA) stimulation experiments. We removed the existing medium from the Petri dish and carefully added 300–500 μL of preheated (at 37°C) magnesium-free extracellular solution. Baseline recordings were made via video microscopy for a duration of 50–200 s. Without pausing the video, 300–500 μL of a mixture containing NMDA (200 μM; Sigma #M3262) and Glycine (20 μM) (Sigma#G7126) was added. An increase in neuronal discharge was observed and recorded for 150–200 s. After introducing 100 μL of MK801 (5 μM) (MCE#HY-15084-10) near the neuron, a gradual decrease in the intensity of neuronal discharge was noted and recorded for 150–200 s.
5
Gelatin Crosslinking and Mechanical Characterization
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A 5% w/v gelatin solution was prepared by dissolving type A gelatin powder (G2500, Sigma-Aldrich, Milan, Italy) in 1x phosphate buffered saline (PBS 1x, Sigma-Aldrich, Milan, Italy) at 50 °C under stirring for 2 h. Cylindrical gelatin samples with flat surfaces were obtained by casting the so-prepared solution in 8 mm height and 13 mm diameter molds, then leaving it to crosslink for 1 h at room temperature (RT). The physically gelled samples were removed from the molds and immersed for 48 h at 4 °C in glutaraldehyde (GTA; G5882, Sigma-Aldrich, Milan, Italy) crosslinking solutions prepared at different concentrations (i.e., 5, 25, 50, and 100 mM) in 40% v/v ethanol water solution, keeping a constant 5:1 volume ratio between the GTA solution and the gelatin samples to crosslink [4 (link)]. After chemical crosslinking, the samples were submerged in 0.5 M glycine solution (G7126, Sigma-Aldrich, Milan, Italy) for 2 h at RT to quench unreacted GTA, then copiously rinsed with deionized water. Finally, the samples were equilibrium swollen in PBS 1x at RT to be in a stable and reproducible state for testing [4 (link),18 (link),20 (link)] (equilibrium weight was reached within 1 h, data not shown), and mechanically characterized as described in the following sections.
6
Gelatin Scaffold Preparation and Crosslinking
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A 5% w/v gelatin solution was prepared by dissolving type A gelatin powder (G2500, Sigma-Aldrich, Milan, Italy) in 1Xphosphate buffered saline (PBS 1X, Sigma-Aldrich) at 50 °C under stirring for 2 h. Cylindrical gelatin samples with ~ 3 mm thickness were obtained by casting 3 mL of the solution into 35 mm diameter Petri dishes (Corning Inc. - Corning, NY, USA) and left to physically crosslink for 2 h at 4 °C. Then, they were exposed to UV light for 20 min (while on ice). Subsequently, 3 mL of glutaraldehyde (GTA; G5882, Sigma-Aldrich) solutions prepared at different concentrations (i.e., 1.25, 2.5, 5, 10, 25, 50 mM) in 40% v/v ethanol/water were added to the samples which were maintained for 48 h at 4 °C for chemical crosslinking. After washing thrice with PBS, samples were incubated in 3 mL of 200 mM glycine solution (G7126, Sigma-Aldrich) in a humidified cell culture incubator (37 °C, 5% CO2) for 1 h to quench unreacted GTA moieties. The samples were then subjected to further rinsing with PBS and ethanol and overnight incubation in cell culture medium (Dulbecco’s Modified Eagles Medium or DMEM, Sigma-Aldrich). Finally, the medium was discarded, and the samples were washed thrice with PBS and readied for mechanical testing (day 0).
7
Immunofluorescent Localization of hnRNP E1
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Cells were cultured to 50–70% confluence on glass-bottom tissue culture dishes (150680; Thermo Fisher Scientific) and then washed with phosphate-buffered solution (PBS, 10010023; Thermo Fisher Scientific). The cells were fixed with 2% paraformaldehyde (FB002; Thermo Fisher Scientific), followed by the addition of glycine buffer (0.1 mM glycine) (G7126; Sigma-Aldrich) for paraformaldehyde quenching and blocked in 1% bovine serum albumin (BSA, TS-38839; Thermo Fisher Scientific) in PBS for 1 h. The hnRNP E1 rabbit polyclonal antibody (sc-28725; Santa Cruz Biotechnology) was diluted in PBS (1:200) with 1% BSA and incubated with the cells at 4°C overnight. The cells were then washed with cold PBS three times prior to incubation with rhodamine-conjugated goat anti-rabbit IgG (1:500; Pierce Biotechnology) for 45 min at room temperature. The labeled cells were washed with cold PBS and imaged using a confocal microscope (Carl Zeiss LSM780; Carl Zeiss Microscopy GmbH, Jena, Germany). The images were analyzed using ZEN 2012 software (Carl Zeiss Microscopy GmbH).
8
Chromatin Immunoprecipitation from Mouse Hippocampus
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Hippocampi from male mice were collected as previously described (Gjoneska et al., 2015 (link)) immediately after euthanasia. ChIP was then performed as described in Gjoneska et al. (2015) (link). In brief, tissues were minced and crosslinked in 1% formaldehyde (28906; Thermo Fisher Scientific) for 15 min at room temperature and quenched with glycine for 5 min (g7126; Sigma-Aldrich). The samples were homogenized in cell lysis buffer containing proteinase inhibitors (C762Q77; Thermo Fisher Scientific) and chromatin was then fragmented to a size range of 200–500 bp using a digital sonifier (SFX 250; Branson). Solubilized chromatin was then diluted and incubated with 1 µg antibody (sc-352x; Santa Cruz) at 4°C overnight. Immune complexes were captured with Protein A sepharose beads (101041; Thermo Fisher Scientific), washed, and eluted. Enriched chromatin was then subjected to crosslink reversal and proteinase K (25530049; Thermo Fisher Scientific) digestion at 65°C, phenol-chloroform (17908; Thermo Fisher Scientific) extraction, and ethanol precipitation.
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