The bromelain assay was established according to Chakraborty et al. (2014) [60 (link)] with some modifications. The reaction involved 50 μL enzyme extract (from PBP) and 1150 μL casein 1% (w/v) (Sigma–Aldrich, Germany) solution in glycine 0.1 mol·L−1 (Sigma–Aldrich, Germany) and 25 mmol·L−1 cysteine (Sigma–Aldrich, Germany). The mixture was incubated in a shaking water bath (10 min at 37 °C) and the reaction was stopped by adding 1.8 mL trichloroacetic acid 5% (w/v). The assay mixture was filtrated with a syringe filter (0.45 μm) and the absorbance measurement was performed at 280 nm. The bromelain activity was calculated using a tyrosine (Alfa Aesar, Lancashire, UK) standard curve and expressed as the amount of tyrosine on a dry weight basis reported as μmol tyrosine. min−1·g−1 dry weight.
Tyrosine
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Tyrosine is a naturally occurring amino acid that plays a vital role in various biological processes. It is an essential component of many proteins and serves as a precursor for the synthesis of important neurotransmitters and hormones. Tyrosine can be utilized in various laboratory applications, such as cell culture media formulation, protein analysis, and biochemical assays. Its core function is to provide a building block for protein synthesis and support cellular functions.
Automatically generated - may contain errors
Lab products found in correlation
Glycine, by Merck Group (2 mentions)
Cysteine, by Merck Group (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Casein, by Merck Group (1 mentions)
Diethylamine, by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Triethylamine, by Merck Group (1 mentions)
Vanillin, by Merck Group (1 mentions)
Acryloyl chloride, by Merck Group (1 mentions)
Tryptophan, by Thermo Fisher Scientific (1 mentions)
Donkey anti mouse alexa 647, by Thermo Fisher Scientific (1 mentions)
Folin ciocalteu reagent, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Glucose oxidase, by Merck Group (1 mentions)
Catalase, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by PAN Biotech (1 mentions)
Purity tu machine, by Avantor (1 mentions)
Cysteamine hydrochloride, by Merck Group (1 mentions)
Guaiacol, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
7 protocols using tyrosine
1
Bromelain Activity Assay Protocol
2
Bromelain Activity Assay Protocol
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The bromelain assay was determined according to Chakraborty et al. (2014) (link) [10] (link) with some modifications reported by Santos et al. (2020) [28] (link). The assay consisted in mixing the enzyme extract (50 µL) and 1150 µL casein 1% (w/v) (Sigma-Aldrich, Germany) solution in 0.1 mol L -1 glycine (Sigma-Aldrich, Germany) and 25 mmol L -1 cysteine. The mixture was incubated in a shaking water bath (10 min at 37 • C) and the reaction was stopped by adding 5% (w/v) trichloroacetic acid (1.8 mL). The assay mixture was filtrated (0.45 µm) and the absorbance was measured at 280 nm. The bromelain activity was calculated using a tyrosine (Alfa Aesar, Lancashine, United Kingdom) standard curve and expressed as the amount of tyrosine on a dry weight basis (µmol tyrosine•min -1 •g -1 of dry weight).
3
Synthesis of Functional Polymer Hydrogels
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Amino acids tyrosine and tryptophan (99% and 97% Acros, Düsseldorf, Germany), (AIBN, 98% Acrōs Germany) 2,2′-azobis(isobutyronitrile) were recrystallized from methanol, and N-isopropylacrylamide (NIPAAm, Düsseldorf) was recrystallized from distilled hexane. Vanillin (99% Düsseldorf, Germany), triethylamine (99% Merck, Darmstadt, Germany), acryloyl chloride (98% Merck, Darmstadt, Germany), formaldehyde (38% Sigma-Aldrich, Darmstadt, Germany), diethyl amine (99% Acros, Düsseldorf, Germany), Dichloromethane, dioxane, tetrahydrofuran (THF), and diethyl ether were distilled over potassium hydroxide. Other chemicals were used as received. For pH 1.68, pH 7 and pH 12.46 buffer solutions were used as purchased from Thermo Fisher (Loughborough, US).
4
Functionalized Polystyrene Particles for Biomolecular Analysis
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Spherical polystyrene latex (SL) particles functionalized with negatively charged sulphate groups and fluorophore conjugated secondary antibody (Donkey anti-mouse Alexa 647) were purchased from Thermo Fischer Scientific. The radius of the SL particles was (215 AE 4) nm, as determined by the manufacturer using transmission electron microscopy in the dried stage. The surface charge density of the particles was reported to be À12 mC m À2 . Papain (PPN) from Carica papaya (3 U mg À1 ), heparin (HEP) sodium salt from porcine intestinal mucosa, Folin & Ciocalteu reagent, anti-HRP primary antibody, glucose oxidase, catalase, phosphate buffered saline (PBS), glucose and cysteamine hydrochloride were purchased from Sigma-Aldrich. Horseradish peroxidase (HRP), Coomassie brilliant blue, hydrogen peroxide, NaCl, NaH 2 PO 4 (anhydrous), Na 2 HPO 4 (anhydrous), orthophosphoric acid (85 w/w%), ethanol, trichloroacetic acid (TCA) and sodium carbonate were bought from WVR, while tyrosine and guaiacol were from Acros Organics. Paraformaldehyde (PFA) was acquired from Alfa Aesar and bovine serum albumin (BSA) was from PanBiotech. These chemicals were of analytical grade and were used as received. All solutions were diluted using ultrapure water obtained from a VWR Purity TU+ machine. The water and the salt solutions were filtered with a 0.1 mm syringe filter (Millex).
5
Rat Hippocampal Neuron Culture Protocol
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The whole study protocol was approved by the Ethics Committee of Nantong University (No. 20080118-001) and all the procedures were followed by the Chinese Guidelines for the Care and Use of Laboratory Animals.
All experiments were implemented on primary cultures of rat hippocampal neurons. Tissues were isolated from embryonic day 18 Sprague-Dawley (SD) rats, and prepared as previously (16 (link)). Briefly, the whole fetal brains were quickly dissected out, and the hippocampi were harvested and digested with 0.25% tyrosine (Gibco, New York, NY, USA) at 37 °C for 5 min. Neurons were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco), and were plated at a density of [6–8]×104 cells/cm2 onto poly-D-lysine-coated 96-well plates or 8 mm glass coverslips in dishes. After 24 hours, the medium was replaced with Neurobasal medium (Gibco) containing 2% B27 supplement (Gibco) and glutamine (0.5 mM, Gibco). After that, half of the medium was replaced with fresh medium every 3 days.
All experiments were implemented on primary cultures of rat hippocampal neurons. Tissues were isolated from embryonic day 18 Sprague-Dawley (SD) rats, and prepared as previously (16 (link)). Briefly, the whole fetal brains were quickly dissected out, and the hippocampi were harvested and digested with 0.25% tyrosine (Gibco, New York, NY, USA) at 37 °C for 5 min. Neurons were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco), and were plated at a density of [6–8]×104 cells/cm2 onto poly-D-lysine-coated 96-well plates or 8 mm glass coverslips in dishes. After 24 hours, the medium was replaced with Neurobasal medium (Gibco) containing 2% B27 supplement (Gibco) and glutamine (0.5 mM, Gibco). After that, half of the medium was replaced with fresh medium every 3 days.
6
Tetrandrine Cytotoxicity and Apoptosis Mechanisms
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Tetrandrine (purity ≥ 98%) was purchased from Aladdin Biochemical Technology Co. (Shanghai, China). Tetrandrine was dissolved in DMSO (Solarbio Science & Technology Co., Beijing, China) and was then added to the cell culture supernatant in appropriate proportions. FBS and tyrosine were obtained from Gibco (Carlsbad, CA, USA). Cell Counting Kit-8 (CCK-8), Hoechst 33258 and Annexin V-PI apoptosis reagents were purchased from Beyotime (Shanghai, China). The TRIzol reagent and qRT-PCR Kit were purchased from Invitrogen (Carlsbad, CA, USA). A cDNA Synthesis Kit was from Takara (Shiga, Japan). The antibodies against Bax, Bcl-2, Akt, p-Akt, and β-actin were purchased from Cell Signalling Technology, Inc. (Danvers, MA, USA). The HRP and FITC goat anti-rabbit antibodies were purchased from Wanleibio Co. (Shenyang, China). The relevant primers were synthesized by Rui Biotech Co. (Qingdao, China).
7
Biochemical Reagents for Cell Culture Experimentation
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Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone Laboratories, Inc. (Logan, UT, USA). 1% penicillin/streptomycin was obtained from GE Healthcare (Chicago, IL, USA). Dimethyl sulfoxide (DMSO), ethanol, lipopolysaccharides (LPS), nitrite assay kit, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), spermidine, histidine, serine, asparagine, glucose, citrulline, glutamate, glucose-6-phosphate, methionine, itaconate, tryptophan and FMOC-glycine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lysine, arginine, aspartate, threonine, glutamine, proline, creatine and tyrosine were purchased from Alfa Aesar (Ward Hill, MA, USA). Apigenin, apigetrin, apiin, bergapten and xanthotoxin were purchased from Toronto Research Chemicals (North York, ON, Canada).
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