Pefabloc sc

Manufactured by Merck Group

Sourced in United States, Germany, Italy

Pefabloc SC is a protease inhibitor that inactivates a broad range of serine proteases. It functions by irreversibly blocking the active site of proteases, preventing their enzymatic activity. Pefabloc SC is commonly used in research and industrial applications to inhibit unwanted proteolytic activity.

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Lab products found in correlation

N octyl β d glucopyranoside, by Merck Group (6 mentions) Nitrocellulose membrane, by GE Healthcare (5 mentions) Bradford assay kit, by Merck Group (3 mentions) Aprotinin, by Merck Group (3 mentions) T per solution, by Thermo Fisher Scientific (3 mentions) R pe streptavidin, by Thermo Fisher Scientific (3 mentions) Leupeptin, by Merck Group (3 mentions) Dpp4 inhibitor, by Merck Group (3 mentions) Pepstatin a, by Merck Group (3 mentions) Bovine thrombin, by Enzyme Research (3 mentions) Formic acid, by Thermo Fisher Scientific (3 mentions) Sterilin petri plastic dishes, by Sarstedt (2 mentions) Papain, by Merck Group (2 mentions) Dnase 1, by Merck Group (2 mentions) Biotin ligase, by Avidity (2 mentions) Enhanced chemi luminescence (ecl), by Roche (2 mentions) Protein g sepharose, by GE Healthcare (2 mentions) Ysi 2700 stat analyzer, by Yellow Springs Instruments (2 mentions) N dodecyl β maltoside, by Merck Group (2 mentions) Sigma p8340 protease inhibitor cocktail, by Roche (2 mentions)

74 protocols using pefabloc sc

Mcm5 Stabilization Optimization

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Example 13

Various stabilisers were tested for tested for their effectiveness in stabilising the Mcm5 samples when combined with TBS-T.

In terms of potential candidates, the 1% FBS (foetal bovine serum), 2.5% BSA (bovine serum albumin), 0.25 mM Pefabloc SC, 0.1 mM Pefabloc SC, 0.5% Sigma P8340 protease inhibitor cocktail and the 0.25× Roche cOmplete protease inhibitor cocktail all had encouraging data without compromising the background or positive sample signal compared to the buffer without stability additives. These data are presented in FIGS. 13A and 13B.

These compounds were tested with various storage temperatures after 6 days to see how they compared to the additive-free TBST and the CytoBuster. These data are presented in FIG. 14.

All the stabilisers were effective at stabilising the compositions. The commercially produced protease inhibitor cocktails (Pefabloc, Sigma P3840 & Roche cOmplete) were stable, but reduced the Mcm5 signal. The 2.5% BSA performed better than the 1% FBS at −20° C. Performance at 4° C. and fresh were equivalent for 1% FBS and 2.5% BSA, and both seem to increase signal comparison to standard TBST buffer. As there was a loss at −20° C. for the BSA, a repeat experiment was performed using additional cell lines to confirm the improvement to stability.

US11391744B2. Methods and kits (2022-07-19). ARQUER DIAGNOSTICS LIMITED [GB]. Inventors: David Nicholas Miller-Jones [GB], Cheryl Nyberg [GB], Ronald Alfred Laskey [GB], Kai Stoeber [GB].

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Reagents and Materials for Cell Stimulation Experiments

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Tissue culture media, antibiotics, fetal calf serum (FCS) and NP40 cell lysis buffer (10×) were purchased from Life Technologies (San Giuliano Milanese, Italy); lipopolysaccaride (LPS) (Ultra-Pure LPS-EB from E. coli 0111:B4 strain), zymosan, Pam3CSK4 (VacciGrade™) and polyinosinic-polycytidylic acid (poly(I:C)) (high molecular weight) were from InvivoGen (Cayla-Invivogen Europe, Toulouse, France); BD CytoFix/CytoPerm and CytoFix were from BD Biosciences (SACCO srl, Cadorago (CO), Italy); Ro-106-9920 (Tocris-Cookson, Space Import–export srl, Milan, Italy); poly-l-lysine hydrobromide (mol wt 70,000–150,000), papain, DNase I (bovine pancreas), trypsin inhibitor, l-leucyl-l-leucine methyl ester (L-LME), SB202190, protease inhibitor cocktail, Pefabloc® SC (100 mM), LPS from E. coli 026:B6 (<5 % protein impurities), polymyxin B and all other biochemicals were purchased from Sigma-Aldrich (Milan, Italy) unless noted otherwise; Falcon tissue culture plasticware was purchased from BD Biosciences. Sterilin petri plastic dishes (10 cm Ø) were obtained from Sarstedt (Verona, Italy).

Marinelli C., Di Liddo R., Facci L., Bertalot T., Conconi M.T., Zusso M., Skaper S.D, & Giusti P. (2015). Ligand engagement of Toll-like receptors regulates their expression in cortical microglia and astrocytes. Journal of Neuroinflammation, 12, 244.

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Oklahoma TTP-HUS Registry: ADAMTS13 Evaluation

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The Oklahoma TTP‐Hemolytic Uremic Syndrome (HUS) Registry is a prospective, population‐based inception cohort of all consecutive patients identified by a request to the Oklahoma Blood Institute (OBI) for plasma exchange treatment for a diagnosis of TTP or HUS. The OBI is the sole provider of plasma exchange treatment for all hospitals in 58 of Oklahoma's 77 counties. Plasma exchange treatment is the standard care for all patients diagnosed with TTP1, 27; therefore, all patients with a suspected initial diagnosis of TTP are enrolled in the Registry.28 Since November 1995, serum samples have been collected immediately before the first plasma exchange, and ADAMTS13 activity levels are tested by two methods.
ADAMTS13 (also known as von Willebrand factor [VWF] cleaving protease) activity was measured by both quantitative immunoblotting of degraded, plasma‐derived VWF substrate and fluorogenic assay using FRETS‐VWF73 substrate, Pefabloc SC (Sigma Aldrich‐catalog #76307). Patients are designated as having severe ADAMTS13 deficiency if ADAMTS13 activity is less than 10% of the ADAMTS13 activity in normal pooled human plasma.2 Patients were diagnosed as having an acquired etiology of TTP if their ADAMTS13 activity was less than 10% activity at the time of their initial episode or at the time of a relapse.29

Terrell D.R., Tolma E.L., Stewart L.M, & Shirley E.A. (2019). Thrombotic thrombocytopenic purpura patients' attitudes toward depression management: A qualitative study. Health Science Reports, 2(11), e136.

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Survivin Expression and Purification Protocol

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Survivin was expressed in E.coli BL21(DE3) star (Merck) cells using the pHIS8 expression vector used by Verdecia et al.^{18 (link)} The expression was induced at OD600 0.6–0.8 in Luria Bertani (LB) media by 0.5 mM IPTG and incubated during 3–4 h at 30 °C. The cells were harvested at 6000 g during 20 min and resuspended in lysis buffer containing 50 mM Tris pH 8, 500 mM NaCl, 10 mM imidazole, 0.25 mM Pefabloc®SC (Sigma), 20 mg/ml DNAse (Sigma) and 0.2 mg/ml Lysozyme (Sigma). The cells were lysed with a high pressure homogenizer, using 3 cycles of 15,000–20,000 bar. Survivin was purified by Ni²⁺ chelation chromatography using 5 ml HisTrap FF column (GE Healthcare). Overnight dialysis in 50 mM Tris pH8, 150 mM NaCl and 1 mM DTT buffer and a gel filtration purification using a Superdex 75 100/300GL column (GE Healthcare) were done before chemical labelling. The SDS PAGE of purified survivin before and after chemical labelling is displayed in Supplementary Fig. S9. The borealin^6–20, hSgol1^291–312 and hSgol2^1066–1085 peptides were chemically synthetized (Genscript).

Garcia-Bonete M.J., Jensen M., Recktenwald C.V., Rocha S., Stadler V., Bokarewa M, & Katona G. (2017). Bayesian Analysis of MicroScale Thermophoresis Data to Quantify Affinity of Protein:Protein Interactions with Human Survivin. Scientific Reports, 7, 16816.

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Recombinant HLA-DRB1*04:01 Tetramer Production

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Recombinant HLA-DRB1*04:01 protein was produced by the BRI Tetramer Core as previously described (56 (link)). Soluble HLA-DRB1*04:01 monomer was purified from insect cell culture supernatants and biotinylated at a sequence-specific site using biotin ligase (Avidity) prior to dialysis into phosphate storage buffer. The biotinylated monomer was loaded with 0.2 mg/mL of peptide by incubating at 37°C for 72 hours in the presence of 2.5 mg/mL n-octyl β-D-glucopyranoside and 1 mM Pefabloc SC (Sigma-Aldrich). Peptide-loaded monomers were conjugated into Tmrs using fluorescently labeled streptavidin (Invitrogen) for 6–18 hours at room temperature at a molar ratio of 8:1.

Song J., Schwenzer A., Wong A., Turcinov S., Rims C., Martinez L.R., Arribas-Layton D., Gerstner C., Muir V.S., Midwood K.S., Malmström V., James E.A, & Buckner J.H. (2021). Shared recognition of citrullinated tenascin-C peptides by T and B cells in rheumatoid arthritis. JCI Insight, 6(5), e145217.

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Simulated Gastrointestinal Digestion Fluids

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The following reagents were used to prepare the simulated digestion fluids: CaCl₂(H₂O), KCl, NaHCO₃, NaCl, MgCl₂(H₂O)₆, NaOH (Merck, Darmstadt, Germany), (NH4)₂CO₃ (Sigma-Aldrich, St. Louis, MO, USA), KH₂PO₄ and HCl (J. T. Baker, Center Valley, PA, USA). The enzymes α-Amylase, pepsin, bile, pancreatin and Pefabloc^® SC were purchased from Sigma-Aldrich^® (St. Louis, MO, USA).

Bettencourt A., Gonçalves L.M., Gramacho A.C., Vieira A., Rolo D., Martins C., Assunção R., Alvito P., Silva M.J, & Louro H. (2020). Analysis of the Characteristics and Cytotoxicity of Titanium Dioxide Nanomaterials Following Simulated In Vitro Digestion. Nanomaterials, 10(8), 1516.

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Western Blot Analysis of Protein Phosphorylation

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The cells were harvested on ice in PhosphoSafe^™ Extraction Reagent and 1 mM Pefabloc^®SC (both Sigma-Aldrich) and lysates were centrifuged at 13,000 rpm and 4 °C for 20 min. The concentration was determined using Bradford assay kit (Sigma-Aldrich). The lysates were then separated by SDS-PAGE gel electrophoresis and transferred to a nitrocellulose membrane (GE Healthcare; Munich, Germany). After blocking for 1h at RT with 5% skim milk in 0.1% TBST (25 mM Tris/HCl pH 7.4; 145 mM NaCl, 2.7 mM KCl, 0.1% Tween 20), the membrane was incubated over night at 4°C with a primary antibody. The β-Actin (1:10000, GeneTex, GTX26276/18985), pERK1/2 (1:200, SantaCruz sc/7383/G0518) and ERK1/2 (1:1000, Cell Signaling 9102/26) antibodies were diluted in 5% skim milk in 0.1% TBST. Next, the blots were washed with 1% TBST and incubated with anti-mouse (Jackson Immuno Research, 115-035-071/ 29454) or anti-rabbit (Jackson Immuno Research, 111-035-046/27362) secondary antibodies in 5% skim milk in 0.1% TBST for an hour at RT. After washing with 0.1% TBST, the bands were visualized with ECL (Roche Diagnostics).

Buchert J., Diederichs S., Kreuser U., Merle C, & Richter W. (2019). The Role of Extracellular Matrix Expression, ERK1/2 Signaling and Cell Cohesiveness for Cartilage Yield from iPSCs. International Journal of Molecular Sciences, 20(17), 4295.

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Prion Protein Digestion and Analysis

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For each digestion experiment, PK (Merck Millipore) was diluted fresh on the day from a glycerol stock to working stock in cell culture grade water. PK working stock concentrations were calculated to digest RT-QuIC reaction products with different amounts of PK. The final digestion volume included 76 μL of sample and 4 μL of a PK working stock solution. Digestions were carried out at 37°C for 1 hour and stopped by placing the tubes on ice. PK activity was irreversibly inhibited by adding Pefabloc SC (Sigma-Aldrich) to 1.25 mM.
Products of PK digestion were methanol precipitated with 10 volumes of -20°C 99.9% pure methanol (Fisher Chemicals). Samples, with added methanol, were thoroughly vortexed and incubated overnight at -20°C. The following day samples were centrifuged for 35 minutes at 18200 x g (Eppendorf 5417R, rotor 06/09 HL128), the supernatant was discarded, and residual methanol evaporated by incubating the open tubes at 100°C in a Class II microbiological safety cabinet. Pellets were resuspended in 42 μL of 0.1%SDS/PBS, half of the volume was moved to a fresh tube and added to an equivalent volume of either LDS4X/2%βMA (reduced samples) or LDS4X with no βMA (not reduced samples).

Piconi G., Peden A.H., Barria M.A, & Green A.J. (2019). Epitope mapping of the protease resistant products of RT-QuIC does not allow the discrimination of sCJD subtypes. PLoS ONE, 14(6), e0218509.

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Recombinant HLA-DRB1*04:01 Tetramer Generation

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HLA-DRB1*04:01 protein used within this study was recombinantly produced in S2 insect cells as previously described [20 ]. All peptides were synthesised at > 95% purity by GenScript (Piscataway, NJ, USA). The biotinylated monomer was loaded with the different peptides (Table 1) by incubation in the presence of n-octyl-β-D-glucopyranoside and Pefabloc SC (Sigma-Aldrich) and subsequently tetramerised using streptavidin conjugated to PE, PE-Cy5, PE-CF594 (BD Biosciences) or APC (Biolegend), respectively. Every single tetramer was loaded with one specific peptide during this individual assembly, so we ended up having ten different tetramers. Three of these were conjugated to PE and PE-CF594, respectively and two conjugated to APC and PE-Cy5, respectively (Table 1).

Gerstner C., Turcinov S., Hensvold A.H., Chemin K., Uchtenhagen H., Ramwadhdoebe T.H., Dubnovitsky A., Kozhukh G., Rönnblom L., Kwok W.W., Achour A., Catrina A.I., van Baarsen L.G, & Malmström V. (2020). Multi-HLA class II tetramer analyses of citrulline-reactive T cells and early treatment response in rheumatoid arthritis. BMC Immunology, 21, 27.

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Generating HLA-DQ Tetramers for Antigen-Specific T Cell Detection

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We made three DQ8 tetramers using biotinylated HLA-DQ monomer provided by (NIH Tetramer Core Facility) using method described by Crawford et al. (Crawford et al., 2011 (link)). Briefly, one tetramer is made of HLA-DQ with x-Id peptide (x-Id tet), one complexed with insulin mimotope (mim-tet) and the third one complexed with CLIP (CLIP-tet). Clip was removed using thrombin and empty monomers were loaded with 0.2 mg/mL of either x-Id or Insulin-mimotope peptide. Loading was conducted at 37°C for 72 h in the presence of 2.5 mg/mL (0.25%) n-octyl-β-D-glucopyranoside and 1 mM Pefabloc SC (Sigma-Aldrich). Peptide-loaded HLA-DQ monomers were tetramerized with PE-conjugated streptavidin (eBioscience) at a molar ratio of 1:4, respectively. HLA-DQ/CLIP monomer was tetramerized as control negative in staining. The successful formation of tetramer complexes were verified by gentle SDS-PAGE. Tetramer staining was performed incubating PBMCs with 2 μg /mL HLA class II tetramer for 1 hr at room temperature in FACS buffer. Antibody specific for surface CD4, TCR and CD19 have been used and samples were acquired. The data was analyzed as described above (Dai et al., 2015 ).

Ahmed R., Omidian Z., Giwa A., Cornwell B., Majety N., Bell D.R., Lee S., Zhang H., Michels A., Desiderio S., Sadegh-Nasseri S., Rabb H., Gritsch S., Suva M.L., Cahan P., Zhou R., Jie C., Donner T, & Hamad A.R. (2019). A Public BCR Present in a Unique Dual-Receptor-Expressing Lymphocyte from Type 1 Diabetes Patients Encodes a Potent T Cell Autoantigen. Cell, 177(6), 1583-1599.e16.

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