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Millipore thrombin
Thrombin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 349 article reviews
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thrombin - by Bioz Stars, 2020-04
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Related Articles

DNA Synthesis:

Article Title: Interleukin-1? and tumour necrosis factor-? modulate airway smooth muscle DNA synthesis by induction of cyclo-oxygenase-2: inhibition by dexamethasone and fluticasone propionate
Article Snippet: The compounds used and their sources were as follows: dexamethasone (9α-fluoro-16α-methyl-prednisolone), essentially fatty acid-free bovine serum albumin fraction V (BSA), L -glutamine, indomethacin, prostaglandin E2 , thrombin (bovine plasma) (Sigma, U.S.A.); amphotericin B (fungizone), human recombinant basic fibroblast growth factor (Promega, U.S.A.); collagenase type CLS 1, elastase (Worthington Biochemical, U.S.A.); dimethyl sulphoxide, foetal calf serum (Flow Laboratories, Australia); Dulbecco's Modified Eagle's Medium (DMEM) (Flow Laboratories, Scotland); Dulbecco `A' phosphate buffered saline (PBS) (Oxoid, U.K.); Monomed A, penicillin-G, versene, streptomycin, trypsin (CSL, Australia); PGE2 antiserum (Upstate Biotechnology Inc., Lake Placid, N.Y., U.S.A.); [6-3 H]-thymidine (5 Ci mmol−1 ), [5,6,8,11,12,14,15 (n)-3 H]PGE2 (183 Ci mmol−1 ) (Amersham, U.K.); emulsifier safe scintillant (Canberra-Packard, Australia); fluticasone propionate (Glaxo Wellcome, U.K.); anti-smooth muscle α-actin (mouse monoclonal) (M851), monoclonal mouse anti-human endothelial, CD 31 (JC/70A) (M823) (Dako Corporation, U.S.A.); anti-smooth muscle myosin (rabbit polyclonal) (Sigma, U.S.A.); rabbit anti-mouse IgG horseradish peroxidase-conjugated, mouse anti-rabbit IgG horseradish peroxidase-conjugated (Silenus Laboratories, Hawthorn, Australia); cyclo-oxygenase-1 (ovine) monoclonal antibody, cyclo-oxygenase-2 (human) monoclonal antibody (Cayman Chemical, Ann Arbor, MI, U.S.A.). .. The highest concentration of vehicle DMSO was 0.01% and the threshold for an action of DMSO on ASM DNA synthesis is 0.3%.

Construct:

Article Title: Backbone NMR assignments of HypF-N under conditions generating toxic and non-toxic oligomers
Article Snippet: Methods and materials 1 H, 13 C, 15 N, isotopically labelled HypF-N was expressed and purified as previously reported (Calloni et al. ), using an N-terminal His-tag construct that was expressed in the E. coli strain M15[PREP4] (Qiagen), grown in M9 minimal media by using 15 N-enriched ammonium chloride and 13 C-enriched glucose, and purified using a nickel column (Sigma Aldrich). .. The HypF-N protein was then cleaved from the nickel resin with thrombin from bovine plasma (Sigma Aldrich) overnight at 4 °C in phosphate buffer.

Transplantation Assay:

Article Title: Exogenous Cytokine-Free Differentiation of Human Pluripotent Stem Cells into Classical Brown Adipocytes
Article Snippet: The 100 µL of fibrin gel was prepared by mixing chilled 89.3 µL aqueous solution (2.8 mg/mL) of fibrinogen from bovine plasma type I-S (F8630, Sigma Chemical Co. LLC), prechilled 6.7 µL collagen type I rat tail (354236, Corning Inc. One Riverfront Plaza, Corning, NY, USA), and prechilled 3 µL aprotinin from bovine lung saline solution (A6279, Sigma Chemical Co. LLC), which was successively added by prechilled 1 µL of 50 U/mL thrombin from bovine plasma lyophilized powder (T4648, Sigma Chemical Co. LLC). .. After 48 to 96 h (for short-term assays) or 9 days (for longer-term assays) from transplantation, mice were anesthetized by using a combination anesthetic (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol).

Incubation:

Article Title: Nuclear transport adapts to varying heat stress in a multistep mechanism
Article Snippet: Flag-Ran (Q69L) and Ran were purified by cleaving off the N-terminal GST tag using thrombin (T7513; Sigma-Aldrich) at 80 U/ml beads. .. Essentially, 25 mM EDTA and 2 mM GTP (for Flag-Q69LRan GTP) or GDP (for Ran GDP) were added to Ran (Q69L) or Ran (WT), and after incubation for 1 h on ice, MgCl2 was added at a final concentration of 50 mM.

Article Title: Optimization of Catheter Based rtPA Thrombolysis in a Novel In Vitro Clot Model for Intracerebral Hemorrhage
Article Snippet: .. In vitro blood clots were produced from 25 mL or 50 mL of human blood supplemented with 10 IE of thrombin (bovine plasma thrombin, Sigma, Germany, final concentration 10 IE/500 μ L) in a balloon tightly closed and incubated 1.5 h in an incubator at 37°C (Heraeus Instruments, Germany). ..

Article Title: Isogenic Pairs of hiPSC-CMs with Hypertrophic Cardiomyopathy/LVNC-Associated ACTC1 E99K Mutation Unveil Differential Functional Deficits
Article Snippet: The CM fibrinogen mix was then quickly mixed with 3 U of thrombin (Sigma #T7513) and pipetted into the aperture between the silicone posts. .. Forming hEHTs were then incubated for 2 hr at 37°C and 7% CO2 and subsequently moved to new 24-well plates filled with DMEM supplemented with 10% horse serum, 10 μg/mL insulin (Sigma #I9278), 33 μg/mL aprotinin (Sigma #A1153), and 1% (v/v) PEST, termed EHT medium.

Article Title: None of the Rotor Residues of F1-ATPase Are Essential for Torque Generation
Article Snippet: .. Cleavage of the β - γ link, when attempted, was done simultaneously with the biotinylation by adding 4 NIH units of thrombin (bovine plasma, Sigma Aldrich, St. Louis, MO) per nmol F1 in the last 10 min of the 30-min incubation. ..

Activity Assay:

Article Title: Exogenous Cytokine-Free Differentiation of Human Pluripotent Stem Cells into Classical Brown Adipocytes
Article Snippet: In Vivo Calorigenic Analyses hESC/hiPSC-derived BAs were subcutaneously transplanted and the calorigenic activity was evaluated by using an infrared camera as previously described [ , ] with following modifications. .. The 100 µL of fibrin gel was prepared by mixing chilled 89.3 µL aqueous solution (2.8 mg/mL) of fibrinogen from bovine plasma type I-S (F8630, Sigma Chemical Co. LLC), prechilled 6.7 µL collagen type I rat tail (354236, Corning Inc. One Riverfront Plaza, Corning, NY, USA), and prechilled 3 µL aprotinin from bovine lung saline solution (A6279, Sigma Chemical Co. LLC), which was successively added by prechilled 1 µL of 50 U/mL thrombin from bovine plasma lyophilized powder (T4648, Sigma Chemical Co. LLC).

Expressing:

Article Title: Nuclear transport adapts to varying heat stress in a multistep mechanism
Article Snippet: Paragraph title: Recombinant protein expression and purification ... Flag-Ran (Q69L) and Ran were purified by cleaving off the N-terminal GST tag using thrombin (T7513; Sigma-Aldrich) at 80 U/ml beads.

Modification:

Article Title: Cell-Type Specific Four-Component Hydrogel
Article Snippet: .. Materials and Methods The following components were employed: pig skin gelatin (GELITA AG, Eberbach, Germany; Lot No. 325109), bacterial enzyme transglutaminase (N-Zyme BioTec GmbH, Darmstadt, Germany Product No. T014, Lot No. 1007T014); fetal calf serum (FCS), Hanks balanced salt solution (HBSS), phosphate buffered saline (PBS), penicillin/streptomycin (pen/strep), gentamycin and L-glutamine (all from PAA, Linz, Austria); DNA/nucleus stain 4,6-diamidino-2-phenylindol (DAPI), human type I fibrinogen from plasma (F3879), laminin (LN), L-ascorbate-2-phospate, nerve growth factor, pig skin gelatin (G2500, Batch# 128K0066), and thrombin from bovine plasma (T4648), FD & C blue #1 (861146) (all from Sigma-Aldrich, Deisenhofen, Germany); calcein (Molecular Probes Inc., USA); poly-D-lysine (PDL) and Dulbecco’s Modified Eagle Medium (DMEM) (BioWhittaker, Verviers, Belgium). .. Vascular endothelial growth factor (VEGF) was from PeproTech EC (recombinant human VEGF165 , London, UK); Matrigel™ (BD Biosciences, USA).

Article Title: A 3D-printed platform for modular neuromuscular motor units
Article Snippet: Materials 3-(Trimethoxysilyl)propyl methacrylate, poly(ethylene glycol) diacrylate, retinoic acid, ciliary neurotrophic factor (CNTF), fibrinogen, thrombin from bovine plasma, aminocaproic acid (ACA), LONG R3 human insulin-like growth factor (IGF-1), Triton X-100, 4′, 6-diamindino-2-phenylindole (DAPI), and L-glutamic acid (glutamate) were obtained from Sigma-Aldrich (St Louis, MO, USA). .. Dulbecco’s modified Eagle’s medium (DMEM), penicillin–streptomycin (10 000 U mL−1 ), and L-glutamine were obtained from Cellgro (Corning, Manassas, VA, USA).

Article Title: A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases
Article Snippet: GST was removed by treatment with bovine plasma thrombin (Sigma), and the lactamases were finally purified by ion exchange through a Sephadex CM-50 column , . .. Bi-Zn(II)-NDM-1 was expressed in a modified version of the pET-28 ( + ) plasmid in which the thrombin cleavage site was replaced by a TEV cleavage site .

Article Title: Interleukin-1? and tumour necrosis factor-? modulate airway smooth muscle DNA synthesis by induction of cyclo-oxygenase-2: inhibition by dexamethasone and fluticasone propionate
Article Snippet: .. The compounds used and their sources were as follows: dexamethasone (9α-fluoro-16α-methyl-prednisolone), essentially fatty acid-free bovine serum albumin fraction V (BSA), L -glutamine, indomethacin, prostaglandin E2 , thrombin (bovine plasma) (Sigma, U.S.A.); amphotericin B (fungizone), human recombinant basic fibroblast growth factor (Promega, U.S.A.); collagenase type CLS 1, elastase (Worthington Biochemical, U.S.A.); dimethyl sulphoxide, foetal calf serum (Flow Laboratories, Australia); Dulbecco's Modified Eagle's Medium (DMEM) (Flow Laboratories, Scotland); Dulbecco `A' phosphate buffered saline (PBS) (Oxoid, U.K.); Monomed A, penicillin-G, versene, streptomycin, trypsin (CSL, Australia); PGE2 antiserum (Upstate Biotechnology Inc., Lake Placid, N.Y., U.S.A.); [6-3 H]-thymidine (5 Ci mmol−1 ), [5,6,8,11,12,14,15 (n)-3 H]PGE2 (183 Ci mmol−1 ) (Amersham, U.K.); emulsifier safe scintillant (Canberra-Packard, Australia); fluticasone propionate (Glaxo Wellcome, U.K.); anti-smooth muscle α-actin (mouse monoclonal) (M851), monoclonal mouse anti-human endothelial, CD 31 (JC/70A) (M823) (Dako Corporation, U.S.A.); anti-smooth muscle myosin (rabbit polyclonal) (Sigma, U.S.A.); rabbit anti-mouse IgG horseradish peroxidase-conjugated, mouse anti-rabbit IgG horseradish peroxidase-conjugated (Silenus Laboratories, Hawthorn, Australia); cyclo-oxygenase-1 (ovine) monoclonal antibody, cyclo-oxygenase-2 (human) monoclonal antibody (Cayman Chemical, Ann Arbor, MI, U.S.A.). .. Stock solutions of dexamethasone (10 m M ) and fluticasone propionate (10 m M ) were prepared in 100% v/v dimethyl sulphoxide (DMSO).

Article Title: Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance
Article Snippet: Protein purification MamM CTD WT and M250L were purified as described previously for WT , with the modification of the Triton-X 100 concentration in buffer A to 0.01% (volume percentage). .. In order to cleave the 6xHis-tag, bovine thrombin (1 U mL−1 ; t4648-10 KU, Sigma-Aldrich) was added to the eluted protein and the mixture was dialyzed for 16 h at 277 K against Buffer E (10 mM Tris-HCl pH = 8, 150 mM NaCl and 5 mM β-mercaptoethanol).

Western Blot:

Article Title: Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance
Article Snippet: In order to cleave the 6xHis-tag, bovine thrombin (1 U mL−1 ; t4648-10 KU, Sigma-Aldrich) was added to the eluted protein and the mixture was dialyzed for 16 h at 277 K against Buffer E (10 mM Tris-HCl pH = 8, 150 mM NaCl and 5 mM β-mercaptoethanol). .. Protein concentration was determined by measuring protein absorption at 280 nm, protein purity was analyzed by SDS-polyacrylamide (20%) gel electrophoresis (PAGE) and protein identification was confirmed by western blot using HRP-conjugated anti-6xHis-tag antibody (ab1187, Abcam, UK) after affinity column and tandem mass spectroscopy.

Crystallization Assay:

Article Title: Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance
Article Snippet: For crystallization of MamM CTD M250L, a fraction of 6xHis-tagged protein was not cleaved after the Ni-NTA affinity purification step (the same protocol was used, but without thrombin). .. In order to cleave the 6xHis-tag, bovine thrombin (1 U mL−1 ; t4648-10 KU, Sigma-Aldrich) was added to the eluted protein and the mixture was dialyzed for 16 h at 277 K against Buffer E (10 mM Tris-HCl pH = 8, 150 mM NaCl and 5 mM β-mercaptoethanol).

Chromatography:

Article Title: A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases
Article Snippet: GST was removed by treatment with bovine plasma thrombin (Sigma), and the lactamases were finally purified by ion exchange through a Sephadex CM-50 column , . .. Mono-Zn(II)-Sfh-I was expressed in a pET-26b plasmid (Novagen) and purified by anion exchange (Q-Sepharose) and size exclusion (Superdex 75) chromatography .

Article Title: Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance
Article Snippet: MamM CTD M250P-expressing cells were suspended in Buffer A (50 mM Tris-HCl pH = 8, 200 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol, 5% glycerol, 0.045% Triton X-100 and 0.03% TWEEN 20) at a weight ratio of 1:2, with DNaseI (10 μg mL−1 ) and a protease inhibitor cocktail (containing phenylmethylsulfonyl fluoride (PMSF), 100 μM; leupeptin, 1.2 μg mL−1 ; and pepstatin A, 1 μM) for 20 min at 277 K. Suspended cells were then disrupted by three cycles of French press pressure cell (Thermo Scientific, NC, US) at 207 MPa and centrifuged at 45,000 RPM (60 Ti fixed angle rotor, Beckman Coulter, CA, US) for 45 min at 277 K. Supernatant fraction was applied to a home-made gravity HIS-Select Cobalt Affinity Gel (5 mL bead volume; H8162, Sigma-Aldrich, Israel) in an Econo-Column (Bio-Rad, CA, US) chromatography column that was pre-equilibrated with buffer A. .. In order to cleave the 6xHis-tag, bovine thrombin (1 U mL−1 ; t4648-10 KU, Sigma-Aldrich) was added to the eluted protein and the mixture was dialyzed for 16 h at 277 K against Buffer E (10 mM Tris-HCl pH = 8, 150 mM NaCl and 5 mM β-mercaptoethanol).

Protease Inhibitor:

Article Title: Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance
Article Snippet: MamM CTD M250P-expressing cells were suspended in Buffer A (50 mM Tris-HCl pH = 8, 200 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol, 5% glycerol, 0.045% Triton X-100 and 0.03% TWEEN 20) at a weight ratio of 1:2, with DNaseI (10 μg mL−1 ) and a protease inhibitor cocktail (containing phenylmethylsulfonyl fluoride (PMSF), 100 μM; leupeptin, 1.2 μg mL−1 ; and pepstatin A, 1 μM) for 20 min at 277 K. Suspended cells were then disrupted by three cycles of French press pressure cell (Thermo Scientific, NC, US) at 207 MPa and centrifuged at 45,000 RPM (60 Ti fixed angle rotor, Beckman Coulter, CA, US) for 45 min at 277 K. Supernatant fraction was applied to a home-made gravity HIS-Select Cobalt Affinity Gel (5 mL bead volume; H8162, Sigma-Aldrich, Israel) in an Econo-Column (Bio-Rad, CA, US) chromatography column that was pre-equilibrated with buffer A. .. In order to cleave the 6xHis-tag, bovine thrombin (1 U mL−1 ; t4648-10 KU, Sigma-Aldrich) was added to the eluted protein and the mixture was dialyzed for 16 h at 277 K against Buffer E (10 mM Tris-HCl pH = 8, 150 mM NaCl and 5 mM β-mercaptoethanol).

Transferring:

Article Title: Exogenous Cytokine-Free Differentiation of Human Pluripotent Stem Cells into Classical Brown Adipocytes
Article Snippet: The 100 µL of fibrin gel was prepared by mixing chilled 89.3 µL aqueous solution (2.8 mg/mL) of fibrinogen from bovine plasma type I-S (F8630, Sigma Chemical Co. LLC), prechilled 6.7 µL collagen type I rat tail (354236, Corning Inc. One Riverfront Plaza, Corning, NY, USA), and prechilled 3 µL aprotinin from bovine lung saline solution (A6279, Sigma Chemical Co. LLC), which was successively added by prechilled 1 µL of 50 U/mL thrombin from bovine plasma lyophilized powder (T4648, Sigma Chemical Co. LLC). .. The 30 µL prechilled fibrin gel or BA-embedded fibrin gel was subcutaneously transplanted in the gluteal region of 5–6 week-old male ICR mice or NOD/ShiJic-scid (NOD/SCID) mice (CLEA Japan, Inc., Tokyo, Japan), whose hair was removed in broad areas around hip using hair removal cream 3 days before, through a 5 mm incised part using a 1000 µL pipette tip.

other:

Article Title: Releasing the Brakes on the Fibrinolytic System in Pulmonary Emboli: Unique Effects of Plasminogen Activation and α2-Antiplasmin Inactivation
Article Snippet: GA); human plasmin (Calbiochem); bovine thrombin (Sigma, St. Louis, MO); citrated frozen human plasma (Lampire Biological Laboratories, PA); chimeric α2-antiplasmin inactivating antibody (TS23, Translational Sciences, Memphis, TN); 125 I-fibrinogen (Perkin-Elmer, MA); FITC-fibrinogen (Molecular Innovations, MI); r-tPA (Alteplase, Genetech Inc.); all the other reagents if not specified (Sigma, St. Louis, MO).

Imaging:

Article Title: A chemical-genetics approach to study the role of atypical Protein Kinase C in Drosophila
Article Snippet: Paragraph title: Live imaging ... The fibrinogen was clotted using thrombin (100 U ml−1 , Sigma-Aldrich, T7513) for 10 min and supplemented Schneider's medium was pipetted on top.

Protein Concentration:

Article Title: Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance
Article Snippet: In order to cleave the 6xHis-tag, bovine thrombin (1 U mL−1 ; t4648-10 KU, Sigma-Aldrich) was added to the eluted protein and the mixture was dialyzed for 16 h at 277 K against Buffer E (10 mM Tris-HCl pH = 8, 150 mM NaCl and 5 mM β-mercaptoethanol). .. Protein concentration was determined by measuring protein absorption at 280 nm, protein purity was analyzed by SDS-polyacrylamide (20%) gel electrophoresis (PAGE) and protein identification was confirmed by western blot using HRP-conjugated anti-6xHis-tag antibody (ab1187, Abcam, UK) after affinity column and tandem mass spectroscopy.

Sequencing:

Article Title: Backbone NMR assignments of HypF-N under conditions generating toxic and non-toxic oligomers
Article Snippet: The HypF-N protein was then cleaved from the nickel resin with thrombin from bovine plasma (Sigma Aldrich) overnight at 4 °C in phosphate buffer. .. Thrombin cleavage generates a non-native N-terminal sequence (Gly-Ser-Ala instead of Met-Ala).

Sonication:

Article Title: Nuclear transport adapts to varying heat stress in a multistep mechanism
Article Snippet: The bacteria pellets were suspended in lysis buffer (50 mM Tris-HCl, pH 8.3, 500 mM NaCl, and 1 mM EDTA, pH 8.0) and lysed by freezing, thawing, and sonication. .. Flag-Ran (Q69L) and Ran were purified by cleaving off the N-terminal GST tag using thrombin (T7513; Sigma-Aldrich) at 80 U/ml beads.

Affinity Purification:

Article Title: Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance
Article Snippet: For crystallization of MamM CTD M250L, a fraction of 6xHis-tagged protein was not cleaved after the Ni-NTA affinity purification step (the same protocol was used, but without thrombin). .. In order to cleave the 6xHis-tag, bovine thrombin (1 U mL−1 ; t4648-10 KU, Sigma-Aldrich) was added to the eluted protein and the mixture was dialyzed for 16 h at 277 K against Buffer E (10 mM Tris-HCl pH = 8, 150 mM NaCl and 5 mM β-mercaptoethanol).

Recombinant:

Article Title: Cell-Type Specific Four-Component Hydrogel
Article Snippet: Materials and Methods The following components were employed: pig skin gelatin (GELITA AG, Eberbach, Germany; Lot No. 325109), bacterial enzyme transglutaminase (N-Zyme BioTec GmbH, Darmstadt, Germany Product No. T014, Lot No. 1007T014); fetal calf serum (FCS), Hanks balanced salt solution (HBSS), phosphate buffered saline (PBS), penicillin/streptomycin (pen/strep), gentamycin and L-glutamine (all from PAA, Linz, Austria); DNA/nucleus stain 4,6-diamidino-2-phenylindol (DAPI), human type I fibrinogen from plasma (F3879), laminin (LN), L-ascorbate-2-phospate, nerve growth factor, pig skin gelatin (G2500, Batch# 128K0066), and thrombin from bovine plasma (T4648), FD & C blue #1 (861146) (all from Sigma-Aldrich, Deisenhofen, Germany); calcein (Molecular Probes Inc., USA); poly-D-lysine (PDL) and Dulbecco’s Modified Eagle Medium (DMEM) (BioWhittaker, Verviers, Belgium). .. Vascular endothelial growth factor (VEGF) was from PeproTech EC (recombinant human VEGF165 , London, UK); Matrigel™ (BD Biosciences, USA).

Article Title: Nuclear transport adapts to varying heat stress in a multistep mechanism
Article Snippet: Paragraph title: Recombinant protein expression and purification ... Flag-Ran (Q69L) and Ran were purified by cleaving off the N-terminal GST tag using thrombin (T7513; Sigma-Aldrich) at 80 U/ml beads.

Article Title: Interleukin-1? and tumour necrosis factor-? modulate airway smooth muscle DNA synthesis by induction of cyclo-oxygenase-2: inhibition by dexamethasone and fluticasone propionate
Article Snippet: .. The compounds used and their sources were as follows: dexamethasone (9α-fluoro-16α-methyl-prednisolone), essentially fatty acid-free bovine serum albumin fraction V (BSA), L -glutamine, indomethacin, prostaglandin E2 , thrombin (bovine plasma) (Sigma, U.S.A.); amphotericin B (fungizone), human recombinant basic fibroblast growth factor (Promega, U.S.A.); collagenase type CLS 1, elastase (Worthington Biochemical, U.S.A.); dimethyl sulphoxide, foetal calf serum (Flow Laboratories, Australia); Dulbecco's Modified Eagle's Medium (DMEM) (Flow Laboratories, Scotland); Dulbecco `A' phosphate buffered saline (PBS) (Oxoid, U.K.); Monomed A, penicillin-G, versene, streptomycin, trypsin (CSL, Australia); PGE2 antiserum (Upstate Biotechnology Inc., Lake Placid, N.Y., U.S.A.); [6-3 H]-thymidine (5 Ci mmol−1 ), [5,6,8,11,12,14,15 (n)-3 H]PGE2 (183 Ci mmol−1 ) (Amersham, U.K.); emulsifier safe scintillant (Canberra-Packard, Australia); fluticasone propionate (Glaxo Wellcome, U.K.); anti-smooth muscle α-actin (mouse monoclonal) (M851), monoclonal mouse anti-human endothelial, CD 31 (JC/70A) (M823) (Dako Corporation, U.S.A.); anti-smooth muscle myosin (rabbit polyclonal) (Sigma, U.S.A.); rabbit anti-mouse IgG horseradish peroxidase-conjugated, mouse anti-rabbit IgG horseradish peroxidase-conjugated (Silenus Laboratories, Hawthorn, Australia); cyclo-oxygenase-1 (ovine) monoclonal antibody, cyclo-oxygenase-2 (human) monoclonal antibody (Cayman Chemical, Ann Arbor, MI, U.S.A.). .. Stock solutions of dexamethasone (10 m M ) and fluticasone propionate (10 m M ) were prepared in 100% v/v dimethyl sulphoxide (DMSO).

Nucleic Acid Electrophoresis:

Article Title: Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance
Article Snippet: In order to cleave the 6xHis-tag, bovine thrombin (1 U mL−1 ; t4648-10 KU, Sigma-Aldrich) was added to the eluted protein and the mixture was dialyzed for 16 h at 277 K against Buffer E (10 mM Tris-HCl pH = 8, 150 mM NaCl and 5 mM β-mercaptoethanol). .. Protein concentration was determined by measuring protein absorption at 280 nm, protein purity was analyzed by SDS-polyacrylamide (20%) gel electrophoresis (PAGE) and protein identification was confirmed by western blot using HRP-conjugated anti-6xHis-tag antibody (ab1187, Abcam, UK) after affinity column and tandem mass spectroscopy.

In Vivo:

Article Title: Exogenous Cytokine-Free Differentiation of Human Pluripotent Stem Cells into Classical Brown Adipocytes
Article Snippet: Paragraph title: 2.6. In Vivo Calorigenic Analyses ... The 100 µL of fibrin gel was prepared by mixing chilled 89.3 µL aqueous solution (2.8 mg/mL) of fibrinogen from bovine plasma type I-S (F8630, Sigma Chemical Co. LLC), prechilled 6.7 µL collagen type I rat tail (354236, Corning Inc. One Riverfront Plaza, Corning, NY, USA), and prechilled 3 µL aprotinin from bovine lung saline solution (A6279, Sigma Chemical Co. LLC), which was successively added by prechilled 1 µL of 50 U/mL thrombin from bovine plasma lyophilized powder (T4648, Sigma Chemical Co. LLC).

Mutagenesis:

Article Title: None of the Rotor Residues of F1-ATPase Are Essential for Torque Generation
Article Snippet: Paragraph title: Purification of mutant TF1 ... Cleavage of the β - γ link, when attempted, was done simultaneously with the biotinylation by adding 4 NIH units of thrombin (bovine plasma, Sigma Aldrich, St. Louis, MO) per nmol F1 in the last 10 min of the 30-min incubation.

Tandem Mass Spectroscopy:

Article Title: Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance
Article Snippet: In order to cleave the 6xHis-tag, bovine thrombin (1 U mL−1 ; t4648-10 KU, Sigma-Aldrich) was added to the eluted protein and the mixture was dialyzed for 16 h at 277 K against Buffer E (10 mM Tris-HCl pH = 8, 150 mM NaCl and 5 mM β-mercaptoethanol). .. Protein concentration was determined by measuring protein absorption at 280 nm, protein purity was analyzed by SDS-polyacrylamide (20%) gel electrophoresis (PAGE) and protein identification was confirmed by western blot using HRP-conjugated anti-6xHis-tag antibody (ab1187, Abcam, UK) after affinity column and tandem mass spectroscopy.

Nickel Column:

Article Title: Backbone NMR assignments of HypF-N under conditions generating toxic and non-toxic oligomers
Article Snippet: Methods and materials 1 H, 13 C, 15 N, isotopically labelled HypF-N was expressed and purified as previously reported (Calloni et al. ), using an N-terminal His-tag construct that was expressed in the E. coli strain M15[PREP4] (Qiagen), grown in M9 minimal media by using 15 N-enriched ammonium chloride and 13 C-enriched glucose, and purified using a nickel column (Sigma Aldrich). .. The HypF-N protein was then cleaved from the nickel resin with thrombin from bovine plasma (Sigma Aldrich) overnight at 4 °C in phosphate buffer.

Microscopy:

Article Title: A chemical-genetics approach to study the role of atypical Protein Kinase C in Drosophila
Article Snippet: They were then imaged on a SP8 confocal microscope (Leica) equipped with a 63× NA 1.2 water immersion objective. .. The fibrinogen was clotted using thrombin (100 U ml−1 , Sigma-Aldrich, T7513) for 10 min and supplemented Schneider's medium was pipetted on top.

Purification:

Article Title: Nuclear transport adapts to varying heat stress in a multistep mechanism
Article Snippet: .. Flag-Ran (Q69L) and Ran were purified by cleaving off the N-terminal GST tag using thrombin (T7513; Sigma-Aldrich) at 80 U/ml beads. .. MBPx2, MBPx2-SV NLS, and MBPx2-CBP80 NLS were eluted by addition of 100 mM maltose monohydrate (136-00612; Wako Pure Chemical Industries) in column buffer (20 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA).

Article Title: A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases
Article Snippet: .. GST was removed by treatment with bovine plasma thrombin (Sigma), and the lactamases were finally purified by ion exchange through a Sephadex CM-50 column , . .. Mono-Zn(II)-Sfh-I was expressed in a pET-26b plasmid (Novagen) and purified by anion exchange (Q-Sepharose) and size exclusion (Superdex 75) chromatography .

Article Title: Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance
Article Snippet: Protein was washed with two washing buffers for further purification: Buffer B (20 mM Tris-HCl pH = 8, 500 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol and 5% glycerol) and Buffer C (20 mM Tris-HCl pH = 8, 150 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol and 5% glycerol) and eluted using Buffer D (20 mM Tris-HCl pH = 8, 150 mM NaCl, 500 mM imidazole, 5 mM β-mercaptoethanol and 5% glycerol). .. In order to cleave the 6xHis-tag, bovine thrombin (1 U mL−1 ; t4648-10 KU, Sigma-Aldrich) was added to the eluted protein and the mixture was dialyzed for 16 h at 277 K against Buffer E (10 mM Tris-HCl pH = 8, 150 mM NaCl and 5 mM β-mercaptoethanol).

Article Title: Backbone NMR assignments of HypF-N under conditions generating toxic and non-toxic oligomers
Article Snippet: Methods and materials 1 H, 13 C, 15 N, isotopically labelled HypF-N was expressed and purified as previously reported (Calloni et al. ), using an N-terminal His-tag construct that was expressed in the E. coli strain M15[PREP4] (Qiagen), grown in M9 minimal media by using 15 N-enriched ammonium chloride and 13 C-enriched glucose, and purified using a nickel column (Sigma Aldrich). .. The HypF-N protein was then cleaved from the nickel resin with thrombin from bovine plasma (Sigma Aldrich) overnight at 4 °C in phosphate buffer.

Article Title: None of the Rotor Residues of F1-ATPase Are Essential for Torque Generation
Article Snippet: Paragraph title: Purification of mutant TF1 ... Cleavage of the β - γ link, when attempted, was done simultaneously with the biotinylation by adding 4 NIH units of thrombin (bovine plasma, Sigma Aldrich, St. Louis, MO) per nmol F1 in the last 10 min of the 30-min incubation.

Protein Purification:

Article Title: Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance
Article Snippet: Paragraph title: Protein purification ... In order to cleave the 6xHis-tag, bovine thrombin (1 U mL−1 ; t4648-10 KU, Sigma-Aldrich) was added to the eluted protein and the mixture was dialyzed for 16 h at 277 K against Buffer E (10 mM Tris-HCl pH = 8, 150 mM NaCl and 5 mM β-mercaptoethanol).

Affinity Column:

Article Title: A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases
Article Snippet: Protein production Mono-Zn(II)-GOB-18 and bi-Zn(II)-BcII were expressed as N-terminal fusions to glutathione-S-transferase (GST) protein and purified by using an affinity column with a glutathione-agarose resin. .. GST was removed by treatment with bovine plasma thrombin (Sigma), and the lactamases were finally purified by ion exchange through a Sephadex CM-50 column , .

Article Title: Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance
Article Snippet: In order to cleave the 6xHis-tag, bovine thrombin (1 U mL−1 ; t4648-10 KU, Sigma-Aldrich) was added to the eluted protein and the mixture was dialyzed for 16 h at 277 K against Buffer E (10 mM Tris-HCl pH = 8, 150 mM NaCl and 5 mM β-mercaptoethanol). .. Protein concentration was determined by measuring protein absorption at 280 nm, protein purity was analyzed by SDS-polyacrylamide (20%) gel electrophoresis (PAGE) and protein identification was confirmed by western blot using HRP-conjugated anti-6xHis-tag antibody (ab1187, Abcam, UK) after affinity column and tandem mass spectroscopy.

Polyacrylamide Gel Electrophoresis:

Article Title: Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance
Article Snippet: In order to cleave the 6xHis-tag, bovine thrombin (1 U mL−1 ; t4648-10 KU, Sigma-Aldrich) was added to the eluted protein and the mixture was dialyzed for 16 h at 277 K against Buffer E (10 mM Tris-HCl pH = 8, 150 mM NaCl and 5 mM β-mercaptoethanol). .. Protein concentration was determined by measuring protein absorption at 280 nm, protein purity was analyzed by SDS-polyacrylamide (20%) gel electrophoresis (PAGE) and protein identification was confirmed by western blot using HRP-conjugated anti-6xHis-tag antibody (ab1187, Abcam, UK) after affinity column and tandem mass spectroscopy.

Lysis:

Article Title: Nuclear transport adapts to varying heat stress in a multistep mechanism
Article Snippet: The bacteria pellets were suspended in lysis buffer (50 mM Tris-HCl, pH 8.3, 500 mM NaCl, and 1 mM EDTA, pH 8.0) and lysed by freezing, thawing, and sonication. .. Flag-Ran (Q69L) and Ran were purified by cleaving off the N-terminal GST tag using thrombin (T7513; Sigma-Aldrich) at 80 U/ml beads.

Mouse Assay:

Article Title: Exogenous Cytokine-Free Differentiation of Human Pluripotent Stem Cells into Classical Brown Adipocytes
Article Snippet: The 100 µL of fibrin gel was prepared by mixing chilled 89.3 µL aqueous solution (2.8 mg/mL) of fibrinogen from bovine plasma type I-S (F8630, Sigma Chemical Co. LLC), prechilled 6.7 µL collagen type I rat tail (354236, Corning Inc. One Riverfront Plaza, Corning, NY, USA), and prechilled 3 µL aprotinin from bovine lung saline solution (A6279, Sigma Chemical Co. LLC), which was successively added by prechilled 1 µL of 50 U/mL thrombin from bovine plasma lyophilized powder (T4648, Sigma Chemical Co. LLC). .. The 30 µL prechilled fibrin gel or BA-embedded fibrin gel was subcutaneously transplanted in the gluteal region of 5–6 week-old male ICR mice or NOD/ShiJic-scid (NOD/SCID) mice (CLEA Japan, Inc., Tokyo, Japan), whose hair was removed in broad areas around hip using hair removal cream 3 days before, through a 5 mm incised part using a 1000 µL pipette tip.

Plasmid Preparation:

Article Title: A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases
Article Snippet: GST was removed by treatment with bovine plasma thrombin (Sigma), and the lactamases were finally purified by ion exchange through a Sephadex CM-50 column , . .. Mono-Zn(II)-Sfh-I was expressed in a pET-26b plasmid (Novagen) and purified by anion exchange (Q-Sepharose) and size exclusion (Superdex 75) chromatography .

Positron Emission Tomography:

Article Title: A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases
Article Snippet: GST was removed by treatment with bovine plasma thrombin (Sigma), and the lactamases were finally purified by ion exchange through a Sephadex CM-50 column , . .. Mono-Zn(II)-Sfh-I was expressed in a pET-26b plasmid (Novagen) and purified by anion exchange (Q-Sepharose) and size exclusion (Superdex 75) chromatography .

In Vitro:

Article Title: Optimization of Catheter Based rtPA Thrombolysis in a Novel In Vitro Clot Model for Intracerebral Hemorrhage
Article Snippet: .. In vitro blood clots were produced from 25 mL or 50 mL of human blood supplemented with 10 IE of thrombin (bovine plasma thrombin, Sigma, Germany, final concentration 10 IE/500 μ L) in a balloon tightly closed and incubated 1.5 h in an incubator at 37°C (Heraeus Instruments, Germany). ..

Spectrophotometry:

Article Title: Elastic Characterization of Transversely Isotropic Soft Materials by Dynamic Shear and Asymmetric Indentation
Article Snippet: The fibrinogen solution was then filtered with a 5 μ m and, subsequently, 0.2 μ m filter, and the concentration was determined by measuring the absorbance at 280 nm with a spectrophotometer. .. Thrombin (Sigma-Aldrich, St. Louis, MO, Product No. T4648) was diluted to 0.4 NIH units/mL with TBS and 50 mM Ca++ .

Produced:

Article Title: Optimization of Catheter Based rtPA Thrombolysis in a Novel In Vitro Clot Model for Intracerebral Hemorrhage
Article Snippet: .. In vitro blood clots were produced from 25 mL or 50 mL of human blood supplemented with 10 IE of thrombin (bovine plasma thrombin, Sigma, Germany, final concentration 10 IE/500 μ L) in a balloon tightly closed and incubated 1.5 h in an incubator at 37°C (Heraeus Instruments, Germany). ..

Article Title: Fibrin Microthreads Support Mesenchymal Stem Cell Growth While Maintaining Differentiation Potential
Article Snippet: Fibrin microthreads were produced according to a previously published protocol. .. Briefly, fibrinogen and thrombin from bovine plasma (Sigma Aldrich, St. Louis, MO) were placed into separate 1 mL syringes.

Concentration Assay:

Article Title: Nuclear transport adapts to varying heat stress in a multistep mechanism
Article Snippet: Flag-Ran (Q69L) and Ran were purified by cleaving off the N-terminal GST tag using thrombin (T7513; Sigma-Aldrich) at 80 U/ml beads. .. Essentially, 25 mM EDTA and 2 mM GTP (for Flag-Q69LRan GTP) or GDP (for Ran GDP) were added to Ran (Q69L) or Ran (WT), and after incubation for 1 h on ice, MgCl2 was added at a final concentration of 50 mM.

Article Title: Optimization of Catheter Based rtPA Thrombolysis in a Novel In Vitro Clot Model for Intracerebral Hemorrhage
Article Snippet: .. In vitro blood clots were produced from 25 mL or 50 mL of human blood supplemented with 10 IE of thrombin (bovine plasma thrombin, Sigma, Germany, final concentration 10 IE/500 μ L) in a balloon tightly closed and incubated 1.5 h in an incubator at 37°C (Heraeus Instruments, Germany). ..

Article Title: A chemical-genetics approach to study the role of atypical Protein Kinase C in Drosophila
Article Snippet: The fibrinogen was clotted using thrombin (100 U ml−1 , Sigma-Aldrich, T7513) for 10 min and supplemented Schneider's medium was pipetted on top. .. 1-NA-PP1 was added to a final concentration of 20 µM during the imaging.

Article Title: Interleukin-1? and tumour necrosis factor-? modulate airway smooth muscle DNA synthesis by induction of cyclo-oxygenase-2: inhibition by dexamethasone and fluticasone propionate
Article Snippet: The compounds used and their sources were as follows: dexamethasone (9α-fluoro-16α-methyl-prednisolone), essentially fatty acid-free bovine serum albumin fraction V (BSA), L -glutamine, indomethacin, prostaglandin E2 , thrombin (bovine plasma) (Sigma, U.S.A.); amphotericin B (fungizone), human recombinant basic fibroblast growth factor (Promega, U.S.A.); collagenase type CLS 1, elastase (Worthington Biochemical, U.S.A.); dimethyl sulphoxide, foetal calf serum (Flow Laboratories, Australia); Dulbecco's Modified Eagle's Medium (DMEM) (Flow Laboratories, Scotland); Dulbecco `A' phosphate buffered saline (PBS) (Oxoid, U.K.); Monomed A, penicillin-G, versene, streptomycin, trypsin (CSL, Australia); PGE2 antiserum (Upstate Biotechnology Inc., Lake Placid, N.Y., U.S.A.); [6-3 H]-thymidine (5 Ci mmol−1 ), [5,6,8,11,12,14,15 (n)-3 H]PGE2 (183 Ci mmol−1 ) (Amersham, U.K.); emulsifier safe scintillant (Canberra-Packard, Australia); fluticasone propionate (Glaxo Wellcome, U.K.); anti-smooth muscle α-actin (mouse monoclonal) (M851), monoclonal mouse anti-human endothelial, CD 31 (JC/70A) (M823) (Dako Corporation, U.S.A.); anti-smooth muscle myosin (rabbit polyclonal) (Sigma, U.S.A.); rabbit anti-mouse IgG horseradish peroxidase-conjugated, mouse anti-rabbit IgG horseradish peroxidase-conjugated (Silenus Laboratories, Hawthorn, Australia); cyclo-oxygenase-1 (ovine) monoclonal antibody, cyclo-oxygenase-2 (human) monoclonal antibody (Cayman Chemical, Ann Arbor, MI, U.S.A.). .. The highest concentration of vehicle DMSO was 0.01% and the threshold for an action of DMSO on ASM DNA synthesis is 0.3%.

Article Title: Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance
Article Snippet: Protein purification MamM CTD WT and M250L were purified as described previously for WT , with the modification of the Triton-X 100 concentration in buffer A to 0.01% (volume percentage). .. In order to cleave the 6xHis-tag, bovine thrombin (1 U mL−1 ; t4648-10 KU, Sigma-Aldrich) was added to the eluted protein and the mixture was dialyzed for 16 h at 277 K against Buffer E (10 mM Tris-HCl pH = 8, 150 mM NaCl and 5 mM β-mercaptoethanol).

Article Title: Elastic Characterization of Transversely Isotropic Soft Materials by Dynamic Shear and Asymmetric Indentation
Article Snippet: The fibrinogen solution was diluted with TBS to a final concentration of 10 mg/mL. .. Thrombin (Sigma-Aldrich, St. Louis, MO, Product No. T4648) was diluted to 0.4 NIH units/mL with TBS and 50 mM Ca++ .

Staining:

Article Title: Cell-Type Specific Four-Component Hydrogel
Article Snippet: .. Materials and Methods The following components were employed: pig skin gelatin (GELITA AG, Eberbach, Germany; Lot No. 325109), bacterial enzyme transglutaminase (N-Zyme BioTec GmbH, Darmstadt, Germany Product No. T014, Lot No. 1007T014); fetal calf serum (FCS), Hanks balanced salt solution (HBSS), phosphate buffered saline (PBS), penicillin/streptomycin (pen/strep), gentamycin and L-glutamine (all from PAA, Linz, Austria); DNA/nucleus stain 4,6-diamidino-2-phenylindol (DAPI), human type I fibrinogen from plasma (F3879), laminin (LN), L-ascorbate-2-phospate, nerve growth factor, pig skin gelatin (G2500, Batch# 128K0066), and thrombin from bovine plasma (T4648), FD & C blue #1 (861146) (all from Sigma-Aldrich, Deisenhofen, Germany); calcein (Molecular Probes Inc., USA); poly-D-lysine (PDL) and Dulbecco’s Modified Eagle Medium (DMEM) (BioWhittaker, Verviers, Belgium). .. Vascular endothelial growth factor (VEGF) was from PeproTech EC (recombinant human VEGF165 , London, UK); Matrigel™ (BD Biosciences, USA).

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  • 99
    Millipore anti thrombin iii
    a , The presence of the three forms of neurocan is not an artifact of storage, concentration, or chondroitinase treatment. The same three bands are seen whether conditioned medium is analyzed before or after storing, concentrating, or chondroitinase treatment. b , Protease inhibitors do not affect neurocan processing. Astrocytes were grown in the presence of <t>aprotinin</t> (1.0 μg/ml), anti-thrombin <t>III</t> (0.1 inhibitor U/ml), or TIMP-2 (0.2 μg/ml) for 4 d. Each inhibitor was added fresh each day. Conditioned medium was concentrated and treated with chondroitinase ABC, and an equal amount of total protein (150 μg) was applied to each lane. The blot was labeled with the 1G2 mAb ( left ) and then relabeled with the 1F6 mAb ( right ). Neither serine protease (aprotinin and anti-thrombin III) nor metalloproteinase (TIMP-2) inhibitors prevented the processing of neurocan.
    Anti Thrombin Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore n terminal sequencing human α thrombin
    Isolation of the C-terminal-derived thrombin peptide FYT21. ( a ) Human <t>α-thrombin</t> was digested with CM from several protease producing (1:PAO1, 2–4: clinical isolates) and non-producing (5: PAOB1; 6: clinical isolate) strains for 1 h at 37 °C and analysed by SDS–PAGE (left) and western blotting (right). Thrombin alone was used as a control (c). The arrow indicates peptides of similar molecular mass as the endogenous HVF18, which are only formed in the presence of CM of elastase producing strains. ( b ) Thrombin was digested with purified P. aeruginosa elastase for various periods of time and analysed using western blotting. Thrombin alone was used as a control directly (C) or after 3 h incubation at 37 °C (C180). ( c ) Thrombin fragments obtained after 1 h digestion, followed by separation by SDS–PAGE, were subjected to N-terminal sequencing. ( d ) HPLC profile of digested thrombin. The insert shows the fractions containing C-terminal epitopes of thrombin. The full sequence of the peptide in fraction 32 was obtained using Orbitrap analysis ( e ). ( f ) A 2D (showing the inter-chain disulphide bond) and a 3D model of thrombin depicting all cleavage sites of elastase. Each colour represents one cleavage site. The C-terminal peptide FYT21 is depicted in red. Of note, the sequences TFGSG and IVEGS represent the N termini of the light and heavy chains, respectively.
    N Terminal Sequencing Human α Thrombin, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore thrombin sensitive r1 nws
    (A) Injection of 200 pmol thrombin-sensing <t>R1-NWs</t> and renal clearance control R2 demonstrated detection of disease by a significant increase in urine R1 concentration ( P = 0.0027) but no significant change in control reporter R2 concentration ( P = 0.30). (B) Injection of 1000-fold lower amounts of thrombin-sensing R1-NWs and control R2 demonstrated a significant increase in disease-sensitive R1 release ( P = 0.017) but no significant change in control reporter R2 ( P = 0.15). (C) Normalization of thrombin-sensitive R1-NW release in diseased mice to control mice revealed an average 2.35-fold increase in R1 signal over 3 orders of magnitude injected dose with a best fit line slope that did not deviate significantly from zero ( P = 0.22).
    Thrombin Sensitive R1 Nws, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    a , The presence of the three forms of neurocan is not an artifact of storage, concentration, or chondroitinase treatment. The same three bands are seen whether conditioned medium is analyzed before or after storing, concentrating, or chondroitinase treatment. b , Protease inhibitors do not affect neurocan processing. Astrocytes were grown in the presence of aprotinin (1.0 μg/ml), anti-thrombin III (0.1 inhibitor U/ml), or TIMP-2 (0.2 μg/ml) for 4 d. Each inhibitor was added fresh each day. Conditioned medium was concentrated and treated with chondroitinase ABC, and an equal amount of total protein (150 μg) was applied to each lane. The blot was labeled with the 1G2 mAb ( left ) and then relabeled with the 1F6 mAb ( right ). Neither serine protease (aprotinin and anti-thrombin III) nor metalloproteinase (TIMP-2) inhibitors prevented the processing of neurocan.

    Journal: The Journal of Neuroscience

    Article Title: Neurocan Is Upregulated in Injured Brain and in Cytokine-Treated Astrocytes

    doi: 10.1523/JNEUROSCI.20-07-02427.2000

    Figure Lengend Snippet: a , The presence of the three forms of neurocan is not an artifact of storage, concentration, or chondroitinase treatment. The same three bands are seen whether conditioned medium is analyzed before or after storing, concentrating, or chondroitinase treatment. b , Protease inhibitors do not affect neurocan processing. Astrocytes were grown in the presence of aprotinin (1.0 μg/ml), anti-thrombin III (0.1 inhibitor U/ml), or TIMP-2 (0.2 μg/ml) for 4 d. Each inhibitor was added fresh each day. Conditioned medium was concentrated and treated with chondroitinase ABC, and an equal amount of total protein (150 μg) was applied to each lane. The blot was labeled with the 1G2 mAb ( left ) and then relabeled with the 1F6 mAb ( right ). Neither serine protease (aprotinin and anti-thrombin III) nor metalloproteinase (TIMP-2) inhibitors prevented the processing of neurocan.

    Article Snippet: To investigate neurocan processing, astrocytes were maintained for 4 d in serum-free DMEM containing one of the following protease inhibitors: anti-thrombin III (0.1 U/ml), aprotinin (1.0 μg/ml), tissue inhibitor of metalloproteinase-2 (TIMP-2, 0.2 μg/ml), leupeptin (1.0 μg/ml), E-64 (1.0 μg/ml; Calbiochem, Nottingham, Notts, UK), cathepsin B inhibitor II (1.0 μg/ml; Calbiochem), or α2 -macroglobulin (10 μg/ml).

    Techniques: Concentration Assay, Labeling

    Isolation of the C-terminal-derived thrombin peptide FYT21. ( a ) Human α-thrombin was digested with CM from several protease producing (1:PAO1, 2–4: clinical isolates) and non-producing (5: PAOB1; 6: clinical isolate) strains for 1 h at 37 °C and analysed by SDS–PAGE (left) and western blotting (right). Thrombin alone was used as a control (c). The arrow indicates peptides of similar molecular mass as the endogenous HVF18, which are only formed in the presence of CM of elastase producing strains. ( b ) Thrombin was digested with purified P. aeruginosa elastase for various periods of time and analysed using western blotting. Thrombin alone was used as a control directly (C) or after 3 h incubation at 37 °C (C180). ( c ) Thrombin fragments obtained after 1 h digestion, followed by separation by SDS–PAGE, were subjected to N-terminal sequencing. ( d ) HPLC profile of digested thrombin. The insert shows the fractions containing C-terminal epitopes of thrombin. The full sequence of the peptide in fraction 32 was obtained using Orbitrap analysis ( e ). ( f ) A 2D (showing the inter-chain disulphide bond) and a 3D model of thrombin depicting all cleavage sites of elastase. Each colour represents one cleavage site. The C-terminal peptide FYT21 is depicted in red. Of note, the sequences TFGSG and IVEGS represent the N termini of the light and heavy chains, respectively.

    Journal: Nature Communications

    Article Title: Pseudomonas aeruginosa elastase cleaves a C-terminal peptide from human thrombin that inhibits host inflammatory responses

    doi: 10.1038/ncomms11567

    Figure Lengend Snippet: Isolation of the C-terminal-derived thrombin peptide FYT21. ( a ) Human α-thrombin was digested with CM from several protease producing (1:PAO1, 2–4: clinical isolates) and non-producing (5: PAOB1; 6: clinical isolate) strains for 1 h at 37 °C and analysed by SDS–PAGE (left) and western blotting (right). Thrombin alone was used as a control (c). The arrow indicates peptides of similar molecular mass as the endogenous HVF18, which are only formed in the presence of CM of elastase producing strains. ( b ) Thrombin was digested with purified P. aeruginosa elastase for various periods of time and analysed using western blotting. Thrombin alone was used as a control directly (C) or after 3 h incubation at 37 °C (C180). ( c ) Thrombin fragments obtained after 1 h digestion, followed by separation by SDS–PAGE, were subjected to N-terminal sequencing. ( d ) HPLC profile of digested thrombin. The insert shows the fractions containing C-terminal epitopes of thrombin. The full sequence of the peptide in fraction 32 was obtained using Orbitrap analysis ( e ). ( f ) A 2D (showing the inter-chain disulphide bond) and a 3D model of thrombin depicting all cleavage sites of elastase. Each colour represents one cleavage site. The C-terminal peptide FYT21 is depicted in red. Of note, the sequences TFGSG and IVEGS represent the N termini of the light and heavy chains, respectively.

    Article Snippet: N-terminal sequencing Human α-thrombin (30 μg) was digested with P. aeruginosa elastase for 1 h, separated on a 10–20% Tris-Tricine gel, blotted onto an Immobilon-pSQ transfer membrane (Millipore, USA) and stained with Coomassie Brilliant blue.

    Techniques: Isolation, Derivative Assay, SDS Page, Western Blot, Purification, Incubation, Sequencing, High Performance Liquid Chromatography

    (A) Injection of 200 pmol thrombin-sensing R1-NWs and renal clearance control R2 demonstrated detection of disease by a significant increase in urine R1 concentration ( P = 0.0027) but no significant change in control reporter R2 concentration ( P = 0.30). (B) Injection of 1000-fold lower amounts of thrombin-sensing R1-NWs and control R2 demonstrated a significant increase in disease-sensitive R1 release ( P = 0.017) but no significant change in control reporter R2 ( P = 0.15). (C) Normalization of thrombin-sensitive R1-NW release in diseased mice to control mice revealed an average 2.35-fold increase in R1 signal over 3 orders of magnitude injected dose with a best fit line slope that did not deviate significantly from zero ( P = 0.22).

    Journal: Journal of the American Chemical Society

    Article Title: Disease Detection by Ultrasensitive Quantification of Microdosed Synthetic Urinary Biomarkers

    doi: 10.1021/ja505676h

    Figure Lengend Snippet: (A) Injection of 200 pmol thrombin-sensing R1-NWs and renal clearance control R2 demonstrated detection of disease by a significant increase in urine R1 concentration ( P = 0.0027) but no significant change in control reporter R2 concentration ( P = 0.30). (B) Injection of 1000-fold lower amounts of thrombin-sensing R1-NWs and control R2 demonstrated a significant increase in disease-sensitive R1 release ( P = 0.017) but no significant change in control reporter R2 ( P = 0.15). (C) Normalization of thrombin-sensitive R1-NW release in diseased mice to control mice revealed an average 2.35-fold increase in R1 signal over 3 orders of magnitude injected dose with a best fit line slope that did not deviate significantly from zero ( P = 0.22).

    Article Snippet: To quantify release kinetics of R1 from thrombin-sensitive R1-NWs, we added physiologic levels of recombinant thrombin (15 nM), purified released reporters by filtration (30 kDa M r Amicon Ultra filters; Millipore), and quantified proteolytically released reporters by ELISA or SiMoA.

    Techniques: Injection, Concentration Assay, Mouse Assay