Protease xiv

Manufactured by Merck Group

Sourced in United States, United Kingdom, Germany

Protease XIV is a laboratory reagent manufactured by Merck Group. It is a proteolytic enzyme that can be used for the digestion and breakdown of protein samples during various biological and biochemical analyses. The core function of Protease XIV is to catalyze the hydrolysis of peptide bonds in proteins.

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Lab products found in correlation

Collagenase 2, by Worthington (5 mentions) Dmem f12, by Thermo Fisher Scientific (5 mentions) Collagenase b, by Roche (4 mentions) Collagenase d, by Roche (4 mentions) Collagenase type 2, by Worthington (4 mentions) Dnase, by Merck Group (3 mentions) Non essential amino acids (neaa), by Merck Group (3 mentions) 2 3 butanedione monoxime, by Merck Group (3 mentions) Hepes, by Quality Biological (3 mentions) Penicillin streptomycin, by Quality Biological (3 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions) L glutamine, by Quality Biological (3 mentions) Chymotrypsin, by Merck Group (3 mentions) Propidium iodide, by Thermo Fisher Scientific (3 mentions) Ciprofloxacin hydrochloride, by Merck Group (2 mentions) Gentamicin sulfate, by Merck Group (2 mentions) Horseradish peroxidase hrp, by Merck Group (2 mentions) Selenocystine, by Merck Group (2 mentions) Selenomethionine, by Merck Group (2 mentions) Fetal bovine serum (fbs), by Merck Group (2 mentions)

Spelling variants of this product

Protease type XIV (116 citations) Protease type XIV from Streptomyces griseus (9 citations) Protease XIV from Streptomyces griseus (7 citations) Protease XIV solution (3 citations) Protease (protease type XIV (2 citations) Bacterial protease type XIV (2 citations) Pronase (Protease XIV, (2 citations) Lyophilized protease XIV powder (2 citations) Protease XIV enzyme (2 citations) Protease type XIV from S. griseus (2 citations)

77 protocols using protease xiv

Enzymatic Extraction for Selenium Speciation

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A one-step enzymatic extraction with protease XIV (Sigma-Aldrich Ltd., Gillingham, Dorset, UK) was applied for selenium speciation. An aliquot of LAB or silage sample (0.1 g DM) was accurately weighed into a 15-ml ultra clean polypropylene centrifuge tube followed by the addition of 20 mg enzyme and 8 ml phosphate buffer (60 mM, pH 7.4). The samples were then capped tightly and put on an automatic shaker for 24 -hs at room temperature. After proteolysis, the samples were centrifuged for 20 min. The supernatants were filtered by 0.45 μm PTFE syringe filters (Sigma-Aldrich Ltd.) and transferred into a clean vial. These solutions were then further diluted by deionised water and analysed by HPLC-UV-HG-AFS using the Millennium Excalibur System (PS Analytical). All Se which was not identified as inorganic (SeVI or SeIV) or organic (seleno-amino acids) was presumed to be Nano-Se as previously proposed by Eszenyi et al. (2011) and Xia et al. (2007) (link). This does over estimate total Nano-Se slightly, but as previously reported when sodium selenite is supplied at greater than 64 mg/l Nano-Se accounts for over 90% of total Se within LAB cultures.

Lee M.R., Fleming H.R., Cogan T., Hodgson C, & Davies D.R. (2019). Assessing the ability of silage lactic acid bacteria to incorporate and transform inorganic selenium within laboratory scale silos. Animal Feed Science and Technology, 253, 125-134.

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Fabrication and Degradation of Silk Fibroin Splints

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Splints were fabricated from reconstituted silk fibroin in a two-step process (Figure 1). First, 150 mg/mL aqueous SF, purified from native Bombyx mori cocoons, was cast into molds and lyophilized to form solid 180° splint constructs (freezing: 0.5°C/min to 40°C, primary drying: −20°C at 100 mTorr, secondary drying: 4°C at 100 mTorr). The splints were then immersed in methanol for 1 hour to render them insoluble. Next, 150 mg/mL SF was spin cast onto the exterior of the splint to a thickness of approximately 1 mm, and the coated splints were immersed in methanol for 60 min. SF splints were sterilized with ethylene oxide and allowed to off-gas for at least 72 hours.
Mechanical properties were assessed with a uniaxial mechanical tester (model 3366, Instron Inc.) as the splints were degraded in a protease solution (1.0 U/mL Protease XIV in phosphate buffered saline (PBS), Sigma-Aldrich) at 37°C to mimic in vivo degradation. Mechanical testing of the hydrated splints comprised of a cyclic compression test (0 to 30%) with 5 cycles at a rate of 1.0 mm/min; the maximum force was extracted from the peak force in the final cycle (N=4 independent samples per time point). Degradation was determined by the change in mass of the dried sample from the dry mass at day 0. Samples were imaged under scanning electron microscopy (SEM) to observe the surface topography as they degraded.

McGill M., Raol N., Gipson K.S., Bowe S.N., Fulk-Logan J., Nourmahnad A., Chung J.Y., Whalen M.J., Kaplan D.L, & Hartnick C.J. (2018). Preclinical assessment of resorbable silk splints for the treatment of pediatric tracheomalacia. The Laryngoscope, 129(9), 2189-2194.

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Biodegradation of Silk Rods Using Protease XIV

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Protease XIV (EC 3.4.24.31, Sigma-Aldrich, Shanghai, China) was used for biodegradation studies as it has been shown to be effective in the digestion of casein as compared to trypsin, chymotrypsin and several other proteases [26 (link)]. Silk rods were incubated in a shaker at 37 °C in a solution of Protease XIV (20 ± 1 mg, pH = 7.4, 3.5 U/mg) and PBS for 28 days. Each specimen contains an approximately equivalent dry mass (1.00 ± 0.02 g) of silk-based rods and each specimen has five replicates. During the degradation, the enzymatic aqueous solutions were replenished weekly. The samples were harvested at different time points followed by rinsing with distilled water 3 times. The residual mass was dried at 60 °C for 24 h to determine the degradation ratio of silk rods.

Yan S., He L., Hai A.M., Hu Z., You R., Zhang Q, & Kaplan D.L. (2023). Controllable Production of Natural Silk Nanofibrils for Reinforcing Silk-Based Orthopedic Screws. Polymers, 15(7), 1645.

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Isolation and Characterization of Silkworm-derived Anticancer Compounds

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Cocoons of Bombyx mori silkworm were procured from Jiangsu Province, China. Sal was provided by the China Institute of Veterinary Drugs Control (Beijing, China). Ptx was provided by the Jiangsu Yew Pharmaceutical Co., Ltd (Jiangsu, China). Ptx injection was provided by the Yangtze River Pharmaceutical Group (Jiangsu, China). Protease XIV (Sigma-Aldrich Co., St Louis, MO, USA), 2,4-dinitrophenyl-hydrazine (Sigma-Aldrich, USA), anti-human/mouse CD44 APC (eBioscience, Inc., San Diego, CA, USA), and anti-mouse CD133 PE (eBioscience Inc.) were used as received. Acetonitrile (EMD Millipore, Billerica, MA, USA) and methanol (EMD Millipore) were of HPLC grade. Milli-Q ultra-pure water was used. Anhydrous calcium chloride, absolute ethanol and other chemicals were of analytical grade and used without further purification. Murine hepatic carcinoma cell line H22 was purchased from the Shanghai Institute of Cell Biology (Shanghai, China).

Wu P., Liu Q., Wang Q., Qian H., Yu L., Liu B, & Li R. (2018). Novel silk fibroin nanoparticles incorporated silk fibroin hydrogel for inhibition of cancer stem cells and tumor growth. International Journal of Nanomedicine, 13, 5405-5418.

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Isolation and Culture of Renal Epithelial Cells

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For RECs isolation from kidney tissues, samples were washed with cold wash buffer (F12 medium containing 5% fetal bovine serum [FBS] and 1% P/S) and minced by sterile scalpel into 0.2–0.5 mm³ sizes to a viscous and homogeneous appearance. The minced tissue was then digested with dissociation buffer including DMEM/F12 (Gibco, USA), 2 mg/ml protease XIV (Sigma, USA), 0.01% trypsin (Gibco, USA) and 10 ng/ml DNase I (Sigma, USA) in 37°C incubator 2 h with gentle agitation. Digested cell suspensions were washed with cold‐wash buffer and passed through 70 μm Nylon mesh (Falcon, USA) to remove aggregates. Cell pellets were collected by centrifuge of 200g and then seeded onto a feeder layer of lethally irradiated 3T3‐J2 cells in modified SCM‐6F8 medium. Human SOX9+ RECs were generated from urine samples and expanded as previously described.²⁹For better visualization of colony growth, td‐Tomato+ RECs derived from mT/mG mice were used. For GFP labelling of cultured SOX9+ RECs, medium containing lentivirus was added to the cell culture together with 10 μg/ml polybrene (1:2000)²⁹ and cell identities were analysed by CytoFLEX LX flow cytometry with appropriate markers before use (SOX9+, ATP1A1− and CDH1−). Analysis was gated using forward and side scatters characteristics and corresponding positive/negative control. Data were analysed using the flow cytometry software, FlowJo (TreeStar).

Nie H., Zhao Z., Zhou D., Li D., Wang Y., Ma Y., Liu X, & Zuo W. (2023). Activated SOX9+ renal epithelial cells promote kidney repair through secreting factors. Cell Proliferation, 56(4), e13394.

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Pharmacological Assays with Key Reagents

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Nystatin, angiotensin II, vasopressin, endothelin, heptanol 18BGRA, collagenase 1A, protease XIV and other chemicals were from Sigma (St Louis, MO). Reagents were thawed and diluted on the day of the experiment and excess discarded daily.

Zhang Z., Payne K, & Pallone T.L. (2016). Descending Vasa Recta Endothelial Membrane Potential Response Requires Pericyte Communication. PLoS ONE, 11(5), e0154948.

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Isolation of Tracheal Epithelial Cells

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Parts of porcine trachea were harvested from pigs euthanized for other purposes at the animal facilities in the University Hospital Aachen. Cell harvesting was approved by the local ethical committee. Cells were isolated according to a protocol first published by Widdicombe et al [9 (link)]. The trachea was split longitudinally cutting through the pars membranacea and opposite to the first cut. Subsequently the mucosa was incised longitudinally. The mucosa stripes were removed and placed into a solution of protease XIV (Sigma, Germany) at 0.4 mg/mL. The stripes were incubated at 4 °C over night. After removal of the stripes and centrifugation (200 g, 5 min) the cells were dispersed in Dulbecco’s Modified Essential Medium (DMEM, Sigma, Germany) containing 10 % fetal calf serum (FCS, PAA, Austria) and plated at 2*10⁴ cells/cm². After 24 h, medium was changed to Gray’s medium, which is a 1:1 mixture of DMEM and LHC-9 (Invitrogen, USA). 1.5 μg/mL of bovine serum albumin were added (Sigma, Germany). When cells reached 70 % of confluence, cells were passaged using 0.5 mg/mL Trypsin/0.22 mg/mL EDTA solution (PAA, Austria) for detachment. Cells from the second passage were used for the experiment.

Albers S., Thiebes A.L., Gessenich K.L., Jockenhoevel S, & Cornelissen C.G. (2016). Differentiation of respiratory epithelium in a 3-dimensional co-culture with fibroblasts embedded in fibrin gel. Multidisciplinary Respiratory Medicine, 11, 6.

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Silk Nanomaterial Compression and Bioactive Incorporation

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Amorphous silk nanomaterials (ASN) was packed into predesigned mold, followed by hot pressing at 632 MPa and variable temperatures (25 °C, 65 °C, 95 °C, 125 °C, 145 °C, 175 °C) for 15 min. After hot pressing, the samples were cooled down to room temperature and used for characterizations. To incorporate bioactive molecules in the system, the silk powder was doped with ciprofloxacin hydrochloride (5 wt%, Sigma-Aldrich), gentamicin sulfate (5 wt%, Sigma-Aldrich), horseradish peroxidase (HRP) (1 wt%, Sigma-Aldrich), and protease XIV (1 wt%, Sigma-Aldrich) by thorough mixing and subject to hot processing.

Guo C., Li C., Vu H.V., Hanna P., Lechtig A., Qiu Y., Mu X., Ling S., Nazarian A., Lin S, & Kaplan D.L. (2019). Thermoplastic Molding of Regenerated Silk. Nature materials, 19(1), 102-108.

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Protease Degradation of Protein Fibers

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The fabricated protein fibers were respectively immersed in PBS buffer and PBS solution with 3.5 U/mL protease XIV (Sigma Aldrich), followed by incubation of at 37 °C in a sterile environment. The mechanical properties of the fibers were measured every 24 h. Data are presented in Supplementary Figs. 14 and 15.

Zhang X., Cui M., Wang S., Han F., Xu P., Teng L., Zhao H., Wang P., Yue G., Zhao Y., Liu G., Li K., Zhang J., Liang X., Zhang Y., Liu Z., Zhong C, & Liu W. (2022). Extensible and self-recoverable proteinaceous materials derived from scallop byssal thread. Nature Communications, 13, 2731.

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Curcumin Nanoparticle Release Assay

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First, 10 mg of curcumin was dissolved in 100 ml of anhydrous ethanol to get a homogenous solution. Then 0.1, 0.2, 0.3, 0.4, and 0.5 mL of the solution was added to anhydrous ethanol to make up to 10 ml. Using anhydrous ethanol as a blank control, the absorbance values were recorded at 435 nm, and a calibration curve was drawn with absorbance (A) as Y-axis and concentration (C) as X-axis. The resulting regression equation: A = 0. 1314C + 0. 0278, r = 0. 9998 (Additional file 1: Fig. S1) (n = 3).
The drug release rate was assessed by protease XIV (Sigma UK) degradation experiments. Cur@SF NP nanoparticles (500 μg curcumin) was incubated in 5 ml of phosphate buffered saline (PBS, pH = 7.4) with protease XIV (1 U/ml at 37 °C). The enzyme solution was changed every two days. The absorbance was recorded at 435 nm, and the release rate was calculated with reference to the calibration curve.

Xu L., Sun Z., Xing Z., Liu Y., Zhao H., Tang Z., Luo Y., Hao S, & Li K. (2022). Cur@SF NPs alleviate Friedreich’s ataxia in a mouse model through synergistic iron chelation and antioxidation. Journal of Nanobiotechnology, 20, 118.

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