Pronase from streptomyces griseus

Manufactured by Merck Group

Sourced in United States, Germany

Pronase is a proteolytic enzyme derived from the bacterium Streptomyces griseus. It is a non-specific protease that hydrolyzes a wide range of proteins and peptides. Pronase is commonly used in research and industrial applications where the digestion of proteins is required.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions) Sodium deoxycholate, by Merck Group (2 mentions) Iodomethane, by Merck Group (2 mentions) Ammonium bicarbonate, by Merck Group (2 mentions) Ammonium acetate, by Merck Group (1 mentions) 5 μm syringe filter, by Merck Group (1 mentions) Influx cell sorter, by BD (1 mentions) β mercaptoethanol, by Merck Group (1 mentions) Trypsin from porcine pancreas, by Merck Group (1 mentions) Papain from carica papaya, by Merck Group (1 mentions) Sorvall lynx 6000, by Thermo Fisher Scientific (1 mentions) α chymotrypsin from bovine pancreas, by Merck Group (1 mentions) Christ gamma 1 20, by Martin Christ (1 mentions) Pronase, by Merck Group (1 mentions) Primaria tissue culture flasks, by BD (1 mentions) Amphotericin b, by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Sodium hydroxide beads, by Merck Group (1 mentions) Acetonitrile, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Pronase (431 citations) Streptomyces griseus (17 citations) Pronase E from Streptomyces griseus (7 citations) Pronase (Streptomyces griseus, (2 citations)

12 protocols using pronase from streptomyces griseus

Comprehensive Carcinoembryonic Antigen Purification

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All the chemicals were of analytical reagent grade
or higher. Proponan-2-ol
(iPrOH), methanol (MeOH), ammonium bicarbonate (ABC), and sodium hydroxide
(NaOH) were purchased from Merck (Darmstadt, Germany). Water (LC-MS
grade), acetonitrile (MeCN) (LC-MS grade), glacial acetic acid (HAc),
hydrochloric acid (HCl), DL-dithiothreitol (DTT), iodoacetamide (IAA),
ammonium acetate (NH₄Ac), formic acid (FA), TPCK-treated
trypsin from bovine pancreas, and Pronase from Streptomyces
griseus were purchased from Sigma-Aldrich (Steinheim, Germany).
Endoproteinase Glu-C from Staphylococcus aureus V8
was supplied by Promega Corporation (Madison, WI, USA). CEA purified
from human colon carcinoma was obtained from MyBioSource, Inc. (CEA1;
San Diego, CA, USA) and Fitzgerald Industries International (CEA2;
Acton, MA, USA). CEA purified from human liver metastases of colorectal
carcinoma cells was obtained from Lee BioSolutions, Inc. (CEA3; Maryland
Heights, MO, USA).

Pont L., Kuzyk V., Benavente F., Sanz-Nebot V., Mayboroda O.A., Wuhrer M, & Lageveen-Kammeijer G.S. (2021). Site-Specific N-Linked Glycosylation Analysis of Human Carcinoembryonic Antigen by Sheathless Capillary Electrophoresis–Tandem Mass Spectrometry. Journal of Proteome Research, 20(3), 1666-1675.

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Synchronized adult C. elegans neuron isolation

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Adult cell isolation was performed as described previously^{18 (link)}. Synchronized day 1 adult CQ574 (lin-15B(n765); jsIs682 [Prab-3::GFP::rab-3 + lin-15(+)]; wqIs3 [Prab3::mCherry]) worms washed with M9 buffer to remove excess bacteria. The pellet (~250 µl) was washed with 500 µl lysis buffer (200 mM DTT, 0.25% SDS, 20 mM Hepes pH 8.0, 3% sucrose) and resuspended in 1000 µl lysis buffer. Worms were incubated in lysis buffer with gentle rocking for 6.5 minutes at room temperature. The pellet was washed 6× with M9 and resuspended in 20 mg/ml pronase from Streptomyces griseus (Sigma-Aldrich). Worms were incubated at room temperature (<20 minutes) with periodic mechanical disruption by pipetting every 2 min. When most worm bodies were dissociated, leaving only small debris and eggs, ice-cold osmo-balanced Leibovitz's L-15 buffer containing 2% fetal bovine serum (Gibco) was added. RNA from FACS-sorted neurons was prepared for RNA-seq and subsequent analysis (see FACS isolation and RNA seq Analysis for more details).

Arey R.N., Kaletsky R, & Murphy C.T. (2019). Nervous system-wide profiling of presynaptic mRNAs reveals regulators of associative memory. Scientific Reports, 9, 20314.

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Neuron Isolation from C. elegans

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Synchronized day 1 adult worms expressing pan-neuronal GFP were used for neuron isolation. Worms were washed with M9 buffer to remove excess bacteria. Worm pellet (~250 μl) was then washed once with 500 μl lysis buffer (200 mM DTT, 0.25% SDS, 20 mM HEPES pH 8.0, 3% sucrose), resuspended in 750 μl lysis buffer and incubated with gentle rocking for 6.5 min at room temperature. Worms were washed 6x with M9 and resuspended in 250 μl of 20 mg ml⁻¹ pronase from Streptomyces griseus (Sigma-Aldrich). Worms were incubated at room temperature (~20 min) with periodic mechanical disruption (100x pipetting every 5 min). When most worm bodies were dissociated, ice-cold PBS buffer containing 2% fetal bovine serum (Life Technologies) was added to stop the digestion reaction. Prior to sorting, cell suspensions were passed through a 5 μm syringe filter (Millipore). Cell suspension were stained with Hoechst and Propidium Iodide for at least 30min. GFP+, Hoechst+ and Propidium Iodide- cells were sorted on a BD Influx cell sorter at the Stanford Shared FACS Facility. As a control, the corresponding GFP-, Hoechst+, and Propidium Iodide- cells were also collected. Cells were directly sorted into RTL buffer from RNAeasy kit. Sorting gates were determined by comparison to day 1 adult wildtype N2 cell suspension without any labels. At least 2000 to 10000 cells were collected per sample.

Maeder C.I., Kim J.I., Liang X., Kaganovsky K., Shen A., Li Q., Li Z., Wang S., Xu X.Z., Li J.B., Xiang Y.K., Ding J.B, & Shen K. (2018). The THO complex coordinates transcripts for synapse development and dopamine neuron survival. Cell, 174(6), 1436-1449.e20.

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Quantification of Carcinogenic BPDE Adducts

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β-Mercaptoethanol (BME, ≥ 99%) was obtained from Sigma-Aldrich (Steinheim, Germany). (±)-anti-, (±)-syn- and (+)-anti-Benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) were purchased from the National Cancer Institute (NCI), Chemical Carcinogen References Standard Repository (Kansas City, KS, USA). Purity of (±)-anti-, (±)-syn- and (+)-anti-BPDE was ca. 95%, 95% and 80%, respectively, determined by derivatisation with BME (cf. Michaud et al.35 (link)). Stock solutions of the three BPDEs (each 10 μg/μL) were prepared in ice-cold tetrahydrofuran (500 μL). Lyophilised human serum albumin (hSA) and pronase from Streptomyces griseus were obtained from Sigma-Aldrich (Steinheim, Germany). All solvents and chemicals used were of HPLC and analytical grade, respectively. Sep-Pak Plus C₁₈ solid phase extraction (SPE) cartridges (360 mg) were purchased from Waters (Milford, MA, USA). Eppendorf Protein LoBind tubes (Hamburg, Germany) were used for albumin samples.
Caution. BPDEs are known carcinogenic compounds. They were handled carefully in a well-ventilated fume-hood and destroyed immediately after use by 1 M aqueous H₂SO₄.

Motwani H.V., Westberg E, & Törnqvist M. (2016). Interaction of benzo[a]pyrene diol epoxide isomers with human serum albumin: Site specific characterisation of adducts and associated kinetics. Scientific Reports, 6, 36243.

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Cell Isolation of Transgenic Worms

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Day 1 adult neuronally GFP-labeled worms (Punc119::GFP or Pmec-4::GFP) were prepared for cell isolation as previously described^{15 (link)} with modifications (Extended Data Fig. 2). Synchronized adult worms were washed with M9 buffer to remove excess bacteria. The pellet (~250 µl) was washed with 500 µl lysis buffer (200 mM DTT, 0.25% SDS, 20 mM Hepes pH 8.0, 3% sucrose) and resuspended in 1000 µl lysis buffer. Worms were incubated in lysis buffer with gentle rocking for 6.5 minutes at room temperature. The pellet was washed 6× with M9 and resuspended in 20 mg/ml pronase from Streptomyces griseus (Sigma-Aldrich). Worms were incubated at room temperature (<20 minutes) with periodic mechanical disruption by pipetting every 2 min. When most worm bodies were dissociated, leaving only small debris and eggs, ice-cold PBS buffer containing 2% fetal bovine serum (Gibco) was added. RNA from FACS-sorted neurons was prepared for RNA-seq and subsequent analysis (see Extended Data for details).

Kaletsky R., Lakhina V., Arey R., Williams A., Landis J., Ashraf J, & Murphy C.T. (2015). The C. elegans adult neuronal IIS/FOXO transcriptome reveals adult phenotype regulators. Nature, 529(7584), 92-96.

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Cell Isolation of Transgenic Worms

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Dromedary Milk Proteolysis Profiles

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Dromedary milk was obtained from a dromedary (Camelus dromedarius) herd belonging to the Livestock and Wildlife laboratory, Arid Lands Institute of Medenine, Tunisia. Fresh dromedary milk was defatted by centrifugation (5000×g, 30 min, 4 °C, centrifuge Sorvall Lynx 6000; Thermo Fisher Scientific, Waltham, MA, USA). Then, it was lyophilized in a freeze dryer (Christ Gamma 1–20; Martin Christ GmbH, Osterode am Harz, Germany) and kept at –20 °C.
Pepsin from porcine stomach mucosa and pancreatin from porcine pancreas were obtained from Bio Basic (Ontario, Canada). Trypsin from porcine pancreas, α-chymotrypsin from bovine pancreas, papain from Carica papaya and pronase from Streptomyces griseus were purchased from Sigma-Aldrich, Merck (St. Louis, MO, USA). All other chemicals and reagents used were of analytical grade.

Oussaief O., Jrad Z., Adt I., Khorchani T, & El-Hatmi H. (2020). Dromedary Milk Protein Hydrolysates Show Enhanced Antioxidant and Functional Properties. Food Technology and Biotechnology, 58(2), 147-158.

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Catalase Activity Stability Assays

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For the catalase activity stability studies, free catalase or catalase-loaded nanoparticles were stir-incubated in PBS with 0.2 wt% pronase (pronase from Streptomyces griseus, Sigma) or in rat serum at 37°C. For the activity stability studies with pronase, at 0h, 1h, 2h, 4h, 8h, and 24h, aliquots were collected, placed on ice, and immediately tested for catalase activity. For the activity stability studies in serum, aliquots were tested at 0h, 0.25h, 1h, 2h, and 6h. Enzyme activities were calculated as the sample activity at a given timepoint divided by the initial sample activity at 0h.

Liao R., Pon J., Chungyoun M, & Nance E. (2020). Enzymatic protection and biocompatibility screening of enzyme-loaded polymeric nanoparticles for neurotherapeutic applications.. Biomaterials, 257, 120238.

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Isolation of Muscle Cells from Beef and Dairy Breeds

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To isolate muscle cells, samples of semitendinosus muscle were thawed immediately in a water bath and subsequently washed in PBS with Penicillinum crystallisatum. After PBS aspiration, the incubation medium (Dulbecco’s modified Eagle medium (DMEM) (Gibco, Life Technologies, Carlsbad, CA, USA), Pronase from Streptomyces griseus (Sigma-Aldrich, Saint Louis, MO, USA) at 0.5 mg/mL, 10% FBS, Penicillinum crystallisatum) was added. Samples were incubated for 1.5 h at 37 °C with constant mixing. Afterwards, the suspension with isolated muscle cells was sieved through a 70-µm nylon filter to separate tissue debris. The filtrate-containing cells were centrifuged three times and resuspended in proliferation medium (10%FBS/DMEM/1% penicillin–streptomycin and 0.5% amphotericin B (Gibco, Life Technologies, Carlsbad, CA, USA)) and transferred to Primaria tissue culture flasks (Becton Dickinson, Franklin Lakes, NJ, USA). The 1-h preplating applied four times finally allowed for achieving 60–70% myoblast purity [16 (link)]. Next, 100,000 cells were transferred to culture flasks. Isolated muscle cells of beef and dairy breeds were cultured in proliferation medium until 80% of confluence, which was changed every 48 h. Then, the cells were trypsinized and centrifuged, the supernatant was discarded, and the cell pellet was frozen for further analysis.

Ciecierska A., Motyl T, & Sadkowski T. (2020). Transcriptomic Profile of Primary Culture of Skeletal Muscle Cells Isolated from Semitendinosus Muscle of Beef and Dairy Bulls. International Journal of Molecular Sciences, 21(13), 4794.

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Proteomic Analysis of Brain and Breast Cancer Cells

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Breast cancer cell line MDA-MB-231BR was generously provided by Dr. Paul Lockman (West Virginia University, School of Pharmacy, Morgantown, WV, USA). Brain cancer cell line CRL-1620, DMEM, EMEM, phosphate-buffered saline (PBS), and fetal bovine serum (FBS) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Trypsin-ethylenediaminetetraacetic acid (EDTA) 1× (25% Trypsin/2.21 mM EDTA), formic acid (FA), HPLC-grade water, methanol, and acetonitrile were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Dimethyl sulfoxide (DMSO), sodium hydroxide beads, iodomethane, pronase from Streptomyces griseus, ammonium bicarbonate (ABC), sodium deoxycholate (SDC), and a mammalian total RNA extraction kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Peptide-N-glycosidase F (PNGase F) and 10 × G7 buffer (0.5 M sodium phosphate), NEBNext rRNA depletion kit (human/mouse/rat), and NEBNext Ultra II directional RNA library prep kit were obtained from New England Biolabs (Ipswich, MA, USA).

Wang J., Peng W., Yu A., Fokar M, & Mechref Y. (2022). Glycome Profiling of Cancer Cell Lines Cultivated in Physiological and Commercial Media. Biomolecules, 12(6), 743.

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