Pefabloc

Manufactured by Merck Group

Sourced in United States, Germany, France, United Kingdom

Pefabloc is a protease inhibitor that is used in laboratory settings to prevent the degradation of proteins during sample preparation and analysis. It acts by inhibiting a broad range of serine proteases, including trypsin, plasmin, and thrombin. Pefabloc is a colorless, crystalline powder that is soluble in water and other aqueous solutions.

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Lab products found in correlation

Protease inhibitor cocktail, by Merck Group (5 mentions) Leupeptin, by Merck Group (5 mentions) Aprotinin, by Merck Group (5 mentions) Pepstatin a, by Merck Group (4 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (3 mentions) Ezrgrt 91k, by Merck Group (3 mentions) Dpp4 inhibitor, by Merck Group (3 mentions) Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (3 mentions) Complete mini protease inhibitor cocktail, by Roche (2 mentions) Phosstop phosphatase inhibitor cocktail, by Roche (2 mentions) Protein inhibitor cocktail, by Merck Group (2 mentions) Ras10 mouse monoclonal primary antibody, by Merck Group (2 mentions) Chemidoc xrs hq system, by Bio-Rad (2 mentions) Quantity one software, by Bio-Rad (2 mentions) Complete edta free protease inhibitor cocktail, by Roche (2 mentions) Digitonin, by Merck Group (2 mentions) Chod iodide test kit, by Roche (2 mentions) Lysozyme, by Merck Group (2 mentions) Streptavidin pe cf594, by BD (2 mentions) Pe cy5 streptavidin, by BD (2 mentions)

Close spelling variants of this product

Pefabloc SC protease inhibitor Pefabloc SC Pefabloc® SC PLUS

89 protocols using pefabloc

Apoptosis Induction by Caspase Inhibitors

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To determine the contributions of different caspases to bacteria-induced apoptosis, hatchling animals were exposed to a series of targeted protease inhibitors. The inhibitors used in this study include the pan-caspase inhibitor z-VAD-FMK, the caspase 8 inhibitor Ac-IETD-CHO, and the caspase 9 inhibitor Ac-LEHD-CMK (Santa Cruz Biotechnology, Dallas, TX) as well as the irreversible serine protease inhibitor, Pefabloc (Sigma Aldrich, St. Louis, MO). All inhibitor solutions were 0.22 µm-filter sterilized and stored at – 80 °C until use. Caspase inhibitors were used at final concentrations of 60 and 100 μM for vial and HARV experiments, respectively. For all experiments, the final concentration of the serine protease inhibitor Pefabloc was 25 μM. These concentrations were chosen in part as they did not cause apparent toxicity to the animal and did not impede colonization. Inhibitor treatment occurred for 2 h before the start of the experiment. Dimethyl sulfoxide (DMSO) controls were run in parallel where appropriate.

Vroom M.M., Troncoso-Garcia A., Duscher A.A, & Foster J.S. (2022). Modeled microgravity alters apoptotic gene expression and caspase activity in the squid-vibrio symbiosis. BMC Microbiology, 22, 202.

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Breast Milk Analysis of Dairy Consumption

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Samples of mature breast milk (40 mL) were obtained through a breast pump equipped with plastic disposable pump sets from 12 non-atopic healthy donors (median age: 32 years old) who delivered at term, 2–4 weeks after parturition. All subjects were from across Campania region in Italy. A written informed consent was provided by the mothers before the milk collection.
Six of the samples were collected from women who drank one cup of pasteurized bovine milk daily and regularly consumed other dairy products. Six control samples were collected from women on a strict milk- and dairy products-free diet since at least one week, opportunely instructed to avoid any possible sources of CMP. Breast milk of milk-drinking mothers was collected 2 hours after a regular milk load (200 mL). A skilled operator at the Department of Translational Medicine of the University of Naples “Federico II” (Italy) supervised the administration of bovine milk, sample collection and code labelling. Within less than 3 min after milk expression, the operator added a serine-protease inhibitor (Pefabloc^®, Sigma, St. Louis, MI, USA) to each sample (1mM final concentration) and immediately froze milk at -20°C. High purity grade solvents and chemicals were from Sigma.

Picariello G., Addeo F., Ferranti P., Nocerino R., Paparo L., Passariello A., Dallas D.C., Robinson R.C., Barile D, & Canani R.B. (2016). Antibody-independent identification of bovine milk-derived peptides in breast-milk. Food & function, 7(8), 3402-3409.

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Cellular Protein Extraction Protocol

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To extract cellular proteins, FL3 and HEK293, cells were harvested, washed twice with ice-cold PBS, and solubilized in lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin) to which complete Mini protease inhibitor cocktail, PhosSTOP phosphatase inhibitor cocktail (both from Roche) and 2.0 mM Pefabloc (Sigma-Aldrich, St Louis, MO, USA) were added immediately before use, on ice for 30 min. Cell lysates were centrifuged at 11,000×g at 4°C to remove cellular debris. The pellets obtained after 400×g, 2,000×g and 15,000×g centrifugation were likewise, after washing and repelleting, solubilized in lysis buffer. Protein content of the cell and vesicle lysates were quantified by Bradford microassays (Bio-Rad, Hercules, CA, USA) with comparison against a BSA standard curve. Values were extrapolated from this fitted curve, with r²>0.99 for all included measurements.

Jeppesen D.K., Hvam M.L., Primdahl-Bengtson B., Boysen A.T., Whitehead B., Dyrskjøt L., Ørntoft T.F., Howard K.A, & Ostenfeld M.S. (2014). Comparative analysis of discrete exosome fractions obtained by differential centrifugation. Journal of Extracellular Vesicles, 3, 10.3402/jev.v3.25011.

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Ras Protein Extraction and Western Blot

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All strains were grown in GMM+YE overnight at 37 °C with agitation at 250 rpm. The resulting fungal mass was filtered and rinsed with sterile, deionized water, and crushed with liquid nitrogen. The macerated hyphal material was resuspended in 1:1 volumes of extraction buffer (25 mM Tris-HCl [pH 7.5], 10mM MgCl₂, 150 mM NaCl, 1 mM EDTA, 0.01% NP-40, 2% glycerol, 1 mM Pefabloc [Sigma], 1 mM protein inhibitor cocktail [Sigma]), and the resulting crude lysates were centrifuged at 3,500 rpm for 9 min at 4°C. Cleared lysates were then transferred to new tubes and the total protein concentration was quantified using the Bradford assay. Fifty milligrams of cleared lysate were boiled before SDS-PAGE separation. Membranes were probed with the anti-Ras, clone RAS10 mouse monoclonal primary antibody (1:2000 dilution, EMD Millipore) and followed by the secondary antibody, a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG2a (1:2000 dilution, Abcam). Blots were imaged using a Bio-Rad ChemiDoc XRS HQ System and QuantityOne software (v4.6.5, Bio-Rad). The assay was performed in biologic triplicate for each strain.

Martin‐Vicente A., Souza A.C., Al Abdallah Q., Ge W, & Fortwendel J.R. (2019). SH3‐class Ras guanine nucleotide exchange factors are essential for Aspergillus fumigatus invasive growth. Cellular Microbiology, 21(6), e13013.

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Purification and Interaction Analysis of GST-Yck3

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GST and GST-Yck3 were expressed in Rosetta (DE3) pLySS cells (Novagen) via induction with 0.1 mM IPTG at 30°C for 5 hours. Cells were resuspended in GST lysis buffer (50mM Tris pH 7.5, 150mM NaCl, 5mM EDTA, 1 mM Pefabloc [Sigma-Aldrich], and cOmplete EDTA-free protease inhibitor cocktail [Roche]) and lysed via sonication. Lysates were clarified via centrifugation at 20,000 g for 30 min at 4°C and incubated with glutathione beads at 4°C with agitation. Immobilized proteins were washed once with GST lysis buffer. Yeast cells grown in yeast extract peptone dextrose at 24°C were resuspended in yeast lysis buffer (125mM NaCl, 50mM NaPO₄ pH 7.4, 10% glycerol, 2mM MgCl₂, 0.5% octyl glucosidase, 1mM DTT, 1× protease inhibitor cocktail (Sigma-Aldrich), and cOmplete EDTA-free protease inhibitor cocktail (Roche)) and lysed with glass beads. GST- and GST-Yck3 bound beads were incubated with clarified yeast cell extracts for 1 hr at 4°C with agitation. Beads were then washed twice with yeast wash buffer (50mM HEPES-KOH pH 7.4, 150mM KCl, 1mM EDTA, 10% glycerol). Bound proteins were analyzed via SDS-PAGE, Gelcode Blue Stain Reagent (Thermo Scientific), and immunoblotting. For input (6.25%), 20 μL lysate was resuspended in 20 μl sample buffer, and 5 μL was loaded into the gel.

Wong S., Hepowit N.L., Port S.A., Yau R.G., Peng Y., Azad N., Habib A., Harpaz N., Schuldiner M., Hughson F.M., MacGurn J.A, & Weisman L.S. (2020). Cargo release from myosin V requires the convergence of parallel pathways that phosphorylate and ubiquitylate the cargo adaptor. Current biology : CB, 30(22), 4399-4412.e7.

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Porcine Pepsin and Pancreatin Characterization

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Porcine pepsin (P7012; 2971 IU/mg), porcine pancreatin (P7545; 6.79 IU/mg), bovine bile extract (B8631; 3.1 mmol/g), and the enzyme inhibitors pepstatin A (P5318) and pefabloc (76,307) were all obtained from Sigma-Aldrich (St. Quentin Fallavier, France). Bovine bile salts concentration and enzyme activities were determined as described in the Electronic Supplementary Information of [39 (link)]. The fluorescent dyes used for Confocal laser scanning microscopy (CLSM) analysis were high-content screening (HCS) LipidTOX™ Green Neutral Lipid Stain (H34475) obtained from Thermo Fisher Scientific (Illkirch, France), Rd-DOPE® Liss Rhod PE 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (810150C), and Fast Green Food Green 3 (FCF) (Sigma F7258, Sigma-Aldrich, St Louis, MO, USA). The standards used for size exclusion chromatography were purchased from Phenomenex (Waters Inc., Milford, MA, USA) (No. ALO-3042 for bovine thyroglobulin, IgA, IgG, ovalbumin and myoglobin) and from Sigma-Aldrich (cytochrome C (C2506) and human angiotensin II (A9525)). All other chemicals were of standard analytical grade.

Le Roux L., Ménard O., Chacon R., Dupont D., Jeantet R., Deglaire A, & Nau F. (2020). Are Faba Bean and Pea Proteins Potential Whey Protein Substitutes in Infant Formulas? An In Vitro Dynamic Digestion Approach. Foods, 9(3), 362.

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PrP Proteinase-K Digestion of Brain Homogenates

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For proteinase-K (PK) digestion of crude brain homogenates, we used protein/enzyme ratios as previous reports [16 (link)]. Briefly, 20 μg of brain homogenate was incubated with 20 μg/mL of PK (New England Biolabs) for 1 h at 37 ⁰C. The reaction was then stopped by addition of Pefabloc (Sigma), and samples were boiled with SDS-sample buffer and electrophoresed on 4–12% NuPAGE gels. For PK digestion of asymmetric flow field-flow fractionation (AF4) fractions, 10 μL of each fraction was incubated with 20 μg/mL of PK in the presence of 20 μg bovine serum albumin (BSA) as the sacrificial substrate to account for the low mass of PrP in eluted fractions, and to assure constant total protein mass in all samples. Reactions were then adjusted to the same volume for all samples and they were incubated with PK for 1 h at 37 ⁰C. The reaction was then stopped by addition of Pefabloc. Samples were then boiled with SDS-sample buffer and separated by SDS-PAGE.

Eskandari-Sedighi G., Cortez L.M., Yang J., Daude N., Shmeit K., Sim V, & Westaway D. (2020). Quaternary Structure Changes for PrPSc Predate PrPC Downregulation and Neuronal Death During Progression of Experimental Scrapie Disease. Molecular Neurobiology, 58(1), 375-390.

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GFAP Quantification in Tissue Samples

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ELISAs were performed to quantify GFAP in tissues as previously described [27 (link)]. Briefly, tissues were homogenized in 3.5 mL (brain hemisphere), 0.4 mL (olfactory bulb, corpus callosum, hippocampus, cortex, cervical spinal cord), or 0.8 mL (brain stem, cerebellum) 2% sodium dodecyl sulfate (SDS), 50 mM Tris (pH 7.4), 5 mM EDTA (pH 7.4), 1X cOmplete Protease Inhibitor Cocktail (Roche Diagnostics Corp.) and 1 mM Pefabloc (Sigma) using a Geno/Grinder, then boiled for 15 min. Total protein concentration was measured using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). For tissues, samples and GFAP standards were diluted in PBS with 0.5% Triton-X and 1% BSA. For sandwich ELISA, plates were coated with SMI-26 (1:1000, anti-GFAP monoclonal cocktail, Covance), blocked with 5% non-fat dry milk, incubated with samples and standards, then incubated with polyclonal rabbit anti-cow GFAP (1:5000; Z0334, Dako), followed by goat anti-rabbit-HRP (1:10,000), then SuperSignal ELISA Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Chemiluminescence was measured on a GloRunner microplate luminometer (Turner Biosystems).

LaPash Daniels C.M., Paffenroth E., Austin E.V., Glebov K., Lewis D., Walter J, & Messing A. (2015). Lithium Decreases Glial Fibrillary Acidic Protein in a Mouse Model of Alexander Disease. PLoS ONE, 10(9), e0138132.

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Lysosome Membrane Permeabilization Assay

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Lysosome membrane permeabilization (LMP) was assessed using methods modified from Aits et al. (2015) (link) and as described previously by our laboratory (Jessop et al., 2017 (link)). BMDM were plated in 24 well plates at a density of 2×10⁵ cells per well. Cells were treated with or without HCQ (25 μM) and with or without cSiO₂ (50 μg/mL). Cells were washed twice with PBS and placed on ice. BMDM were then incubated with 200 μL of cytosol extraction buffer, which consisted of 250 mM sucrose, 20 mM Hepes, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 0.5 mM pefabloc (Sigma-Aldrich cat. 76307-100mg), pH 7.5, and digitonin (15 μg/mL ) (Sigma-Aldrich cat. D141-100MG), for 15 min on ice with rocking. The concentration of digitonin for optimal extraction of the cytosolic fraction was determined by titration. β-N-acetylglucosaminidase (NAG) activity was measured by adding 30 μL cytosolic extract to 100 μL of NAG reaction buffer (0.2 M sodium citrate, pH 4.5 with 300 μg/mL 4-methylumbelliferyl-2-acetamido-2-deoxy-β-D-glucopyranoside (Sigma-Aldrich cat. 37067-30-4) and assessed on a plate reader (20 min; 45s intervals; 356 nm excitation; 444 nm emission). Extracted cytosolic LDH activity was measured as described above and used as a control to which the NAG activities were normalized.

Burmeister R., Rhoderick J.F, & Holian A. (2019). Prevention of Crystalline Silica-Induced Inflammation by the Anti-malarial Hydroxychloroquine. Inhalation toxicology, 31(7), 274-284.

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Rb Protein Expression Analysis by Western Blot

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Cells were lysed with RIPA buffer containing Pefabloc (Sigma-Aldrich, Saint Louis, MO, USA). Lysates (100 µg) were mixed with sample buffer, denatured and loaded in Mini-Protean TGX precast gel (Bio-Rad) for SDS-PAGE and transferred to PVDF membranes (GE Healthcare, Chicago, IL, USA). Membranes were blocked with 3% ECL prime blocking agent (GE Healthcare) before primary antibodies (1:1000) against retinoblastoma protein (Rb (D20, Cat#9313S) or Phospho-Rb (Ser-795, Cat#9301S), Cell Signaling Technology, Danvers, MA, USA) were added. Membranes were washed with phosphate buffer saline (PBS)/0.05% Tween 20 before anti-rabbit IgG (1:9000, Cat#ab97051, Abcam, Cambridge, UK) was added. ECL prime western blotting detection reagents (GE Healthcare) were used for development, and bands were detected by ChemiDoc Touch Imaging system and analyzed with Image Lab 6 (Bio-Rad).

Wenthe J., Naseri S., Labani-Motlagh A., Enblad G., Wikström K.I., Eriksson E., Loskog A, & Lövgren T. (2021). Boosting CAR T-cell responses in lymphoma by simultaneous targeting of CD40/4-1BB using oncolytic viral gene therapy. Cancer Immunology, Immunotherapy, 70(10), 2851-2865.

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