The amidolytic activity of tPA was measured spectroscopically using the chromogenic substrate S-2288™ (Chromogenix, Nr. 8205239, USA). Freshly prepared tPA stock solution (1 mg/mL in water; Actilyse Cathflo, Boehringer Ingelheim, Germany) was diluted to 0.1 mgtPA/mL. The tPA working solution (0.1 mgtPA/mL) were diluted 1:20, 1:16.6, and 1:13.3, respectively. SPION stock solutions (3 mgFe/mL) and the supernatants of the SPION preparations were diluted to 1:16.6 and 1:1.6, respectively using sterile deionized H2O. Fifty microliters of diluted tPA and SPION samples were pipetted in quadruplicates into a 96-well cell culture plate mixed with 100 μL assay buffer (1 mM S-2288 in 0.1 M Tris-HCl pH 8.4). The kinetic of the reaction were recorded by measuring the absorption at 405 nm in a spectrophotometer (FilterMax F5, Molecular Devices, Sunnyvale, CA, USA) overnight. To compare SPION-containing and SPION-free samples, the data of SPION-containing samples were subtracted with the value given by the difference in the starting absorption of samples with SPIONs and control samples.
S 2288
Manufactured by Werfen
Sourced in Italy, United States
The S-2288 is a laboratory equipment product manufactured by Werfen. It is designed for automated blood coagulation analysis. The core function of the S-2288 is to perform quantitative measurements of coagulation parameters in patient samples.
Automatically generated - may contain errors
Lab products found in correlation
Filtermax f5, by Molecular Devices (2 mentions)
Versamax microplate reader, by Molecular Devices (2 mentions)
M 2245, by Bachem (2 mentions)
S 2366, by Werfen (2 mentions)
S 2222, by Werfen (2 mentions)
S 2238, by Werfen (2 mentions)
S 2302, by Werfen (2 mentions)
S 2765, by Werfen (2 mentions)
Infinite m200, by Tecan (2 mentions)
Rox reference dye, by Thermo Fisher Scientific (1 mentions)
Pd98059, by Merck Group (1 mentions)
Anti actin 1 19, by Santa Cruz Biotechnology (1 mentions)
Pierce phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Anti p p38, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
May gr nwald eosine methylene blue solution, by Merck Group (1 mentions)
Giemsa azur eosine methylene blue solution, by Merck Group (1 mentions)
Sybr greener supermix, by Thermo Fisher Scientific (1 mentions)
Anti p jnk, by Cell Signaling Technology (1 mentions)
16 protocols using s 2288
1
Measuring tPA Activity with SPIONs
2
Quantifying Mast Cell Enzyme Activities
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For tryptase activity, the chromogenic substrate S-2288 (Chromogenix, Milano, Italy) was used, and M-2245 (N-(4-methoxyphenylazoformyl)-Phe-OH) (Bachem, Bubendorf, Switzerland) was used to detect CPA3 activity. One half million cells were lysed with 150 μl lysis buffer (2M NaCl and 0.5% Triton-X 100 in PBS) for half an hour on ice. Ten μl lysate was then mixed with 90 μl autoclaved water, followed by adding 20 μl of the respective substrate (from stock solutions in H2O: 10 mM S-2288, 1.8 mM M-2245). The activity was monitored by reading the absorbance at 405 nm over 30 min using a Versa max microplate reader (Molecular Devices, Sunnyvale, CA, USA). Each measurement was performed in triplicates or quadruplicates.
3
Inhibitor and Antibody Characterization Protocol
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PD98059 (MEK inhibitor) was obtained from Sigma-Aldrich (Product number P215). U0126 (CAS109511-58-2) (MEK inhibitor) was purchased from EMD Millipore (Burlington, MA, USA). For both inhibitors, stock solutions were prepared in DMSO. The following antibodies were used: anti-ERK1/2, anti-P-ERK1/2, anti-P-JNK and anti-P-P38 [Cell Signaling Technology, anti-actin (I-19); Santa Cruz Biotechnology, Dallas, TX, USA], and anti-Mcpt6 antiserum (raised in rabbits). Monocloncal IgE anti-Dinitrophenyl (IgE anti-DNP) antibody was from Sigma (Stockholm, Sweden; product number: D8406). CellTiter-Blue Cell Viablity Assay was purchased from Promega-Invitrogen (Madison, WI, USA). The chromogenic substrate S-2288 was obtained from Chromogenix (Milan, Italy). May-Grünwald Eosine-methylene blue solution (product number: HX68862424) and Giemsa Azur-Eosine-methylene blue solution (product number: HX128350) were from Merck KGaA (Darmstadt, Germany). SYBR GreenER SuperMix and Rox reference dye were from Invitrogen (Carlsbad, CA, USA). Pierce phosphatase inhibitor was obtained from Thermo Fisher Scientific (Waltham, MA, USA) and protease inhibitor cocktail was from Roche Diagnostics.
4
Protease Activity Measurement Protocols
5
Characterization of Haemachatus haemachatus Venom
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Lyophilized H. haemachatus crude venom was purchased from South African Venom Suppliers (Louis Trichardt, South Africa). Reagents for thromboplastin time, thrombin time and activated partial thromboplastin time (APTT) were from Helena Laboratories (Beaumont, Texas, USA). Reagents for N-terminal sequencing were from Applied Biosystems (Carlsbad, California, USA). The chromogenic substrates, S-2222, S-2288, S-2238, S-2251, S-2444, S-2366 and S-2302 were from Chromogenix (Milano, Italy). Spectrozyme FIXa was from American Diagnostica Inc (Stamford, Connecticut, USA). Superdex 30 HiLoad (16/60) column and Jupiter C18 (5 μ, 300 Å, 4.6 × 250 mm) were purchased from GE Healthcare (Uppsala, Sweden) and Phenomenex (Torrance, California, USA), respectively. All other chemicals and reagents used were of the highest purity.
6
Comprehensive Coagulation Factor Assay Protocol
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Serine proteases factor XIa (FXIa), factor IXa (FIXa), factor VIIa (FVIIa), factor X (FX), factor Xa (FXa) and Russell’s viper venom-X activator (RVV-X) were obtained from Haematologic Technologies Inc. (Vermont, USA), factor XIIa (FXIIa) was procured from Merck Calbiochem (Darmstadt, Germany), factor VIII (FVIII) from Creative Biomart (NY, USA), phospholipid blend from Avanti Polar Lipids Inc. (Alabama, USA) and tissue factor Innovin from Siemens (Murburg, Germany). The chromogenic substrates namely spectrozyme (MeSO2-D-CHG-Gly-Arg-pNA.AcOH) was purchased from Sekisui Diagnostics (MA, USA), rest of the substrates like S-2366 (pyroGlu-Pro-Arg-pNA•HCl), S-2302 (H-D-Pro-Phe-Arg-pNA•2HCl), S-2222 (Bz-IIe-Glu(γ-OR)-Gly-Arg-pNA•HCl), S-2765 (Z-D-Arg-Gly-Arg-pNA•2HCl)S-2238 (H-D-Phe-Pip-Arg-pNA•2HCl) and S-2288 (H-D-Ile-Pro-Arg-pNA•2HCl) were procured from Chromogenix (NJ,USA).
7
Enzymatic Activity Measurements: Optimized Protocols
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Active site titration was performed as described earlier21 . The enzyme was buffer-exchanged into 5 mM Tris (pH 8.0), 150 mM NaCl, 2 mM CaCl2. 50 µM of p-nitrophenyl p’-guanidinobenzoate (PNPGB) in methanol was used as substrate and the burst was measured at 402 nm using 18300 M−1 cm−1 as extinction coefficient. FSAP activity assays were performed as described previously22 (link). In brief, 96 well microtiter plates were used and the standard assay system consisted of 25 mM Tris (pH 7.4), 137 mM NaCl, with CaCl2 (2 mM) and 0.1% (wt/vol) Tween-20 and 0.25 mM of the chromogenic substrate S-2288 (H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilinedihydro-chloride) (Chromogenix, Mölndal, Sweden) and was followed over a period of 60 min at 37 °C at 405 nm in a microplate reader. The data was fitted to the Michaelis-Menten equation using Graphpad Prism software, Version 7.02 (San Diego, CA).
8
Effect of Glycosaminoglycans on Corin Proteolysis
9
Kinetic Analysis of FX Activation and Inhibition
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Peptide substrate hydrolysis (S-2288; Chromogenix), competitive inhibition by pABA, chemical modification of the N-terminal I153 (15 (link), 20 (link)), and inhibition by the zymogen conformation-specific antibody F3-3.2a (10 (link)) was conducted essentially as described elsewhere. Activation of FX and des-gla FX in solution and on phospholipid vesicles (10:90 PS:PC) were carried out as detailed in refs. 15 (link) and 20 (link). Kinetics of inhibition by human plasma-derived AT was determined under pseudofirst-order conditions in the presence of low molecular weight heparin, as described elsewhere (33 (link)). See SI Materials and Methods for detailed experimental protocols.
10
Purification and Characterization of FVIIa Variants
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The FVIIa variants (FVIIaDVQ, FVIIaDV, FVIIaDQ and FVIIaVQ) were expressed and purified as described previously,4 , 9 , 10 FVIIaDVQ was inhibited by incubation with D‐Phe‐Phe‐Arg chloromethylketone (fFR‐ck),11 and mAb4F5 was generated by standard hybridoma technology after immunizing mice with FVIIaDVQ. To produce the Fab fragment of mAb4F5 (Fab4F5), the antibody was digested with papain in 15 mmol/L cysteine with 2 mmol/L EDTA for 6 hours at 37°C at an enzyme‐to‐antibody ratio of 1:100 (w/w). Fab4F5 was purified from the digestion by protein A affinity chromatography in phosphate‐buffered saline followed by cation‐exchange chromatography (GE Healthcare MonoS 5/50 GL) in 25 mmol/L acetate, pH 5.5.
Purified Fab4F5 was dialyzed in 20 mmol/L Tris‐HCl, pH 7.4, overnight and concentrated to about 10 mg/mL using stirred ultrafiltration cells (Millipore and Amicon Bioseparations, Model‐5124) for crystallization. The purity and homogeneity of the protein were confirmed by reduced SDS‐PAGE and analytical gel filtration.
The enzymatic activity of 20 nmol/L FVIIaDVQ in the absence and presence of 100 nmol/L mAb4F5 was measured in 50 mmol/L HEPES, pH 7.4, containing 0.1 mol/L NaCl, 5 mmol/L CaCl2, 0.1% (w/v) bovine serum albumin, using 1 mmol/L S‐2288 (Chromogenix, Milan, Italy).
Purified Fab4F5 was dialyzed in 20 mmol/L Tris‐HCl, pH 7.4, overnight and concentrated to about 10 mg/mL using stirred ultrafiltration cells (Millipore and Amicon Bioseparations, Model‐5124) for crystallization. The purity and homogeneity of the protein were confirmed by reduced SDS‐PAGE and analytical gel filtration.
The enzymatic activity of 20 nmol/L FVIIaDVQ in the absence and presence of 100 nmol/L mAb4F5 was measured in 50 mmol/L HEPES, pH 7.4, containing 0.1 mol/L NaCl, 5 mmol/L CaCl2, 0.1% (w/v) bovine serum albumin, using 1 mmol/L S‐2288 (Chromogenix, Milan, Italy).
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