Pefabloc sc plus

Manufactured by Merck Group

Sourced in Germany

Pefabloc® SC PLUS is a serine protease inhibitor developed by Merck Group. It is designed to inhibit a wide range of serine proteases. The product is intended for laboratory use only.

Automatically generated - may contain errors

Lab products found in correlation

Ezgra 88 k kit, by Merck Group (3 mentions) Bradford solution, by Bio-Rad (2 mentions) K562 nk cell lines, by Thermo Fisher Scientific (2 mentions) Competitive radioimmunoassay, by Merck Group (2 mentions) Inactivated fetal bovine serum, by Thermo Fisher Scientific (1 mentions) Antimycin, by Merck Group (1 mentions) Oligomycin, by Merck Group (1 mentions) C2759, by MedChemExpress (1 mentions) Pei 25000, by Polysciences (1 mentions) C4859, by Merck Group (1 mentions) Turbofectin, by OriGene (1 mentions) Tunicamycin, by MedChemExpress (1 mentions) Valinomycin, by Bio-Techne (1 mentions) Urea, by Bio-Rad (1 mentions) Ammonium bicarbonate, by Merck Group (1 mentions) Np 40, by Thermo Fisher Scientific (1 mentions) Hydrochloric acid (hcl), by Merck Group (1 mentions) Sucrose, by Merck Group (1 mentions)

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Pefabloc SC Pefabloc

8 protocols using pefabloc sc plus

ARTC2.2-Mediated Protein ADP-Ribosylation

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In vitro modification of proteins from K562 cell extracts by ARTC2.2 recombinant protein was performed as described (Palazzo et al., 2016 (link)). Briefly, 8 × 10⁶ cells were washed twice in PBS and lysed in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Triton X‐100, 0.2 mM DTT, 1 μM Olaparib, 4 mM Pefabloc SC PLUS (Sigma‐Aldrich) at 4°C. The cell extract was clarified by centrifugation and diluted five times in no-Triton X‐100 buffer and supplemented with 15 mM MgCl₂ and 1 μCi (37 kBq) of ³²P-NAD⁺. 67 μL extract aliquots were then incubated with or without 1 μM recombinant mARTC2.2 protein for 15 min at 30°C. After 15 min incubation, lysates were further incubated in presence of hydrolases for 45 min at 30°C. Samples were then analysed by SDS-PAGE and autoradiography.

Fontana P., Bonfiglio J.J., Palazzo L., Bartlett E., Matic I, & Ahel I. (2017). Serine ADP-ribosylation reversal by the hydrolase ARH3. eLife, 6, e28533.

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Quantitative PARP Activity Assay

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K562 NK cell lines (ATCC) were cultured in RPMI‐1640 (+L‐Glutamine) supplemented with 10% inactivated foetal bovine serum (Life Technology, Thermo Fisher Scientific) and 1% penicillin/streptomycin. About 8 × 10⁶ cells were washed twice in PBS and then lysed 20 min in 50 mm Tris/HCl pH 7.5, 150 mm NaCl, 0.5% Triton X‐100, 0.2 mm DTT, 1 μm Olaparib, 4 mm Pefabloc^® SC PLUS (Sigma‐Aldrich) at 4 °C. After centrifugation at 15 900 g for 20 min, proteins in cell extract were quantified using Bradford solution (Biorad) and 100 μg·μL⁻¹ BSA standard diluted to 1 μg·μL⁻¹ into lysis buffer. Lysate was diluted five times with no‐Triton X‐100 buffer up to 0.6 μg·μL⁻¹ protein concentrations. Of diluted lysate, 1 mL was supplemented with 1 μCi (37 kBq) of [³²P]‐labelled NAD⁺ and 15 mm MgCl₂. Exactly, 67‐μL extract aliquots were then incubated or not with 1 μm recombinant mARTC2.2 for 15 min at 30 °C. After 15‐min incubation, lysates were additionally incubated or not with several concentrations of NUDT16 and mENPP2‐1‐T for 45 min at 30 °C. Loading sample buffer was added, samples boiled for 4 min at 90 °C and 30 μL was fractionated on SDS/PAGE.

Palazzo L., Daniels C.M., Nettleship J.E., Rahman N., McPherson R.L., Ong S., Kato K., Nureki O., Leung A.K, & Ahel I. (2016). ENPP1 processes protein ADP‐ribosylation in vitro. The Febs Journal, 283(18), 3371-3388.

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Characterization of ARTC2-dependent PARylation

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K562 NK cell lines (ATCC) were cultured in RPMI-1640 (+L-Glutamine) supplemented with 10% Inactivated Fetal Bovine Serum (Life technology) and 1% Penicillin/Streptomycin. 8×10⁶ cells were washed twice in PBS and then lysed 20 minutes in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 0.2 mM DTT, 1μM Olaparib, 4 mM Pefabloc® SC PLUS (Sigma-Aldrich) at 4°C. After centrifugation at 13000 rpm for 20 minutes, proteins in cell extract were quantified using Bradford solution (Biorad) and 100 μg/μl BSA standard diluted to 1 μg/μl into lysis buffer. Lysate was diluted 5-times with no-Triton X-100 buffer up to 0.6 μg/μl protein concentrations. 1 ml of diluted lysate was supplemented with 1 μCi of [³²P]-labelled NAD⁺ and 15 mM MgCl₂. 67 μl extract aliquots were then incubated or not with 1 μM recombinant mARTC2.2 for 15 minutes at 30°C. After 15 minutes incubation, lysates were additionally incubated or not with several concentrations of NUDT16 and mENPP2-1-T for 45 minutes at 30°C. Loading sample buffer was added, samples boiled for 4 minutes at 90°C and 30 μl fractionated on SDS-PAGE.

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Mitochondrial Membrane Potential Assessment

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Cells were treated with CCCP (Sigma-Aldrich, C2759, 20 μM unless stated otherwise), CDDO (MedChemExpress, HY-14909, 2.5 μM), tunicamycin (MedChemExpress, HY-A0098, 10 μM), GTPP (GTPP hexafluorophosphate, MedChemExpress, HY-102007A, 10 μM), oligomycin (CST, 9996L, 10 μM or 25 μM), antimycin (Sigma-Aldrich, A8674, 50 μM), valinomycin (BioTechne, 3373, 0.5 μM), pepstatin A (Enzo, ALX-260-085-M005, 50 μM), 1,10-phenanthroline monohydrate (Sigma-Aldrich, P9375, 500 μM), E-64 (Sigma-Aldrich, E3132, 1 μM), E-64d (Enzo, BML-PI107-0001, 50 μM), zVAD (Z-VADFMK, PeptaNova, 3188-v, 20 μM), 3,4-dichloroisocoumarin (Sigma-Aldrich, D7910, 100 μM), Pefabloc SC Plus (Sigma-Aldrich, 1187360100, 500 μM), ISRIB (Sigma-Aldrich, SML0843, 200 nM), haemin (Sigma-Aldrich, 51280, 20 μM) or cycloheximide (Sigma-Aldrich, C4859, 20 μg ml⁻¹) for the indicated times.
Where indicated, cells were transfected 24 to 36 h before treatment using polyethylenimine (PEI 25000, Polysciences) or Turbofectin (OriGene Technologies). Transductions were performed as described previously^{26 (link)}.
Mitochondrial membrane potential was assessed using 100 nM tetramethylrhodamine (TMRM; Thermo Fisher Scientific) following the manufacturer’s instructions.

Fessler E., Eckl E.M., Schmitt S., Mancilla I.A., Meyer-Bender M.F., Hanf M., Philippou-Massier J., Krebs S., Zischka H, & Jae L.T. (2020). A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol. Nature, 579(7799), 433-437.

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Proteomic Analysis of Cellular Lysates

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For proteomic analysis, cells were washed with PBS and lysed in homogenized buffer [0.3 M sucrose (Merck, Darmstadt, Germany), 0.5 M Tris (AMRESCO, Solon, OH, USA)/HCl (Merck), and 1.67 mM Pefabloc SC PLUS (Merck)]. After centrifugation at 12,000× g for 5 min, the supernatant was collected and ultracentrifugated at 30,000× g for 1 h at 4 °C to remove CNT and impurities. Collected supernatants were then dialyzed against 100 mM ammonium bicarbonate (Sigma) for 10 h at 4 °C. Protein concentration was determined by the modified Bradford assay using BSA as the standard [51 (link)]. A total of 400 μg soluble proteins were lyophilized and solubilized in 175 μL lysis buffer [9.5 M urea (Bio-Rad Laboratories, Hercules, CA, USA), 2% NP-40 (USB Corporation, Cleveland, OH, USA), 2% v/v Ampholyte 3–10 (Serva Electroresis GmbH, Heidelberg, Germany), and 65 mM dithiothreitol (USB Corporation)] for protein analysis.

Li Y.Z., Cheng C.S., Chen C.J., Li Z.L., Lin Y.T., Chen S.E, & Huang S.Y. (2014). Functional Annotation of Proteomic Data from Chicken Heterophils and Macrophages Induced by Carbon Nanotube Exposure. International Journal of Molecular Sciences, 15(5), 8372-8392.

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Measuring Plasma Ghrelin Levels

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To measure plasma ghrelin concentrations, 2 mL EDTA tubes were prepared with 100 μL protease inhibitor (Pefabloc^® SC Plus, Merck KGaA, Germany) before blood collection. All blood samples were put on ice immediately after venepuncture, then centrifuged for 15 min at 4°C and 3,200 g. Samples were then aliquoted and ghrelin plasma was stabilized with HCl before storage at -80°C in in-house freezer facilities. Ghrelin samples were analyzed at the Hormone Laboratory at Oslo University Hospital (Oslo, Norway). Active (acylated) ghrelin concentrations were determined using the EZGRA-88 K kit (Merck, Germany) in duplicates (total analytical CV at 488 pg/mL 12%).

Sailer U., Riva F., Lieberz J., Campbell-Meiklejohn D., Scheele D, & Pfabigan D.M. (2023). Hungry for compliments? Ghrelin is not associated with neural responses to social rewards or their pleasantness. Frontiers in Psychiatry, 14, 1104305.

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Ghrelin and Adiponectin Biomarker Analysis

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Blood samples for ghrelin assessment were collected in 2 ml EDTA tubes that were prepared with 100 μl protease inhibitor (Pefabloc® SC Plus, Merck KGaA, Germany). Immediately afterwards, the blood samples were centrifuged for 15 min at 4 • C and 3200g. The blood plasma was then aliquoted and stabilized with HCl. Ghrelin plasma was then frozen at -80 • C until analysis at the Hormone laboratory at Oslo University Hospital. Active (acylated) ghrelin concentrations were determined using the EZGRA-88 K kit (Merck, Germany) in duplicates (total analytical CV at 488 pg/ml 12 %).
Blood samples for adiponectin assessment were collected in 2 ml EDTA tubes that were centrifuged with the same parameters as the ghrelin samples. Afterwards, blood plasma was aliquoted and frozen at -80 • C until analysis at the Hormone Laboratory at Oslo University Hospital. Adiponectin was analysed with competitive radio immuno assay (Merck Millipore, Germany) in duplicates (total analytical CV at 1300 nmol/l 29 %).

Pfabigan D.M., Vezzani C., Thorsby P.M, & Sailer U. (2022). Sex difference in human olfactory sensitivity is associated with plasma adiponectin. Hormones and behavior, 145.

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Blood Biomarker Sampling and Analysis

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Blood samples for ghrelin assessment were collected in 2 ml EDTA tubes that were prepared with 100µl protease inhibitor (Pefabloc® SC Plus, Merck KGaA, Germany). Immediately afterwards, the blood samples were centrifuged for 15 minutes at 4⁰C and 3200 g. The blood plasma was then aliquoted and stabilized with HCl. Ghrelin plasma was then frozen at -80⁰C until analysis at the Hormone laboratory at Oslo University Hospital. Active (acylated) ghrelin concentrations were determined using the EZGRA-88K kit (Merck, Germany) in duplicates (total analytical CV at 488 pg/ml 12%).
Blood samples for adiponectin assessment were collected in 2ml EDTA tubes that were centrifuged with the same parameters as the ghrelin samples. Afterwards, blood plasma was aliquoted and frozen at -80⁰C until analysis at the Hormone Laboratory at Oslo University Hospital. Adiponectin was analysed with competitive radio immuno assay (Merck Millipore, Germany) in duplicates (total analytical CV at 1300 nmol/l 29%).

Pfabigan D.M., Vezzani C., Thorsby P.M., & Sailer U. (2022). Sex difference in human olfactory sensitivity is associated with plasma adiponectin.

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