Protac and the fluorogenic peptide substrate Pefafluor PCa (Pyroglu-Pro-Arg-AMC) were obtained from Pentapharm (Basel, Switzerland). Human TM and the peptide Gly-Pro-Arg-Pro were purchased from Sigma-Aldrich (Saint Louis, USA). Human α-thrombin was from Haematologic Technologies (Essex Junction, USA) and obtained from CellSystems (St. Katharinen, Germany). Aprotinin was purchased from PanReac AppliChem ITW Reagents (Darmstadt, Germany). Bivalirudin (Angiox®) was obtained from The Medicines Company (Oxfordshire, UK). The biotinylated APC-binding aptamer HS02-52G was synthesized and purified by Microsynth (Balgach, Switzerland).
Gly pro arg pro
Manufactured by Merck Group
Sourced in United States, Spain
Gly-Pro-Arg-Pro is a peptide sequence that can be used in various laboratory applications. It serves as a core functional component for specific assays and experiments, but a detailed description of its intended use would require further information to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Human α thrombin, by Prolytix (1 mentions)
Oxypurinol, by Merck Group (1 mentions)
Human thrombin, by Merck Group (1 mentions)
Ng nitro l arginine methyl ester l name, by Merck Group (1 mentions)
Adenosine 5 diphosphate adp, by Merck Group (1 mentions)
Sodium nitrite nano2, by Merck Group (1 mentions)
Fitc labeled pac1, by BD (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Fam devd fmk, by Merck Group (1 mentions)
Fam letd fmk, by Merck Group (1 mentions)
Fam lehd fmk, by Merck Group (1 mentions)
Annexin 5 fitc, by Merck Group (1 mentions)
Fitc labelled annexin 5, by BD (1 mentions)
Annexin 5, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
4 protocols using gly pro arg pro
1
Fluorogenic Assay for Thrombin Activity
2
Platelet Activation Assay Protocol
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Sodium nitrite (NaNO2), adenosine 5′ diphosphate (ADP), collagen, human thrombin, NG-Nitro-L-Arginine Methyl Ester (L-NAME), Gly-Pro-Arg-Pro (GPRP), oxypurinol and paraformaldehyde were purchased from Sigma (St.Louis, MO). Diethylamine diazeniumdiolate (DEANONOate) was purchased from Cayman chemical (Ann Arbor, MI), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (C-PTIO) was purchased from Alexis (Lausen, Switzerland). FITC-labeled PAC1, PE-labeled anti-human CD62P, PE-Cy5 anti-human CD41a and FITC- or PE-conjugated isotypic MoAbs, and flow cytometry graded PBS were purchased from Becton Dickinson (BD; San Jose, CA).
3
Platelet Phosphatidylserine Exposure and Caspase Activation
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We assessed the surface exposure of phosphatidylserine in washed platelets by measuring the binding of FITC-labelled annexin V (BD Pharmingen, Madrid, Spain). Briefly, washed platelets were resuspended in annexin V binding buffer (10 mM HEPES, 10 mM sodium hydroxide, 140 mM sodium chloride, 2.5 mM calcium chloride, pH 7.4) and labelled with FITC-annexin V. After a 15-min incubation period with either buffer or 1 µM ionomycin (Sigma, Madrid, Spain) at room temperature in the dark, the samples were analysed by flow cytometry.
To analyse active caspase-3, -8 or -9, we diluted PRP 10-fold with isotonic HEPES buffered saline with calcium ion (150 mM sodium chloride, 2 mM calcium chloride, 2 mM magnesium chloride, 2 mM HEPES, pH 7.4), 2 mM Gly-Pro-Arg-Pro (SIGMA, Madrid, Spain) and either FAM-DEVD-FMK, FAM-LETD-FMK or FAM-LEHD-FMK (Millipore, Madrid, Spain).
To analyse active caspase-3, -8 or -9, we diluted PRP 10-fold with isotonic HEPES buffered saline with calcium ion (150 mM sodium chloride, 2 mM calcium chloride, 2 mM magnesium chloride, 2 mM HEPES, pH 7.4), 2 mM Gly-Pro-Arg-Pro (SIGMA, Madrid, Spain) and either FAM-DEVD-FMK, FAM-LETD-FMK or FAM-LEHD-FMK (Millipore, Madrid, Spain).
4
Plasma and Serum Killing Assay
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The killing activity of pooled fresh and heated plasma, fresh and heated serum and, alternatively, plasma treated with the tetrapeptide Gly-Pro-Arg-Pro (Sigma-Aldrich) to prevent clotting (50 μg/mL final concentration), was evaluated in 96 well plates. In all experiments, plasma and serum were obtained from the same group of fish.
Plasma and serum were heated to 45 o C for 30 minutes. Heating the plasma and serum inactivates complement and also the coagulation capacity of plasma. In some experiments, we used pooled plasma and serum from 5 immunized fish. The experiment was carried out under aseptic conditions. Ten µl of ciliates (250 ciliates in 3.5 % NaCl), 9 µl of several dilutions of pooled fresh and heated serum or plasma in 3.5% NaCl, 5 µl of 3.5 % NaCl, and 15 µl of 40 mM CaCl 2 in distilled water were added to each well (total 39 µl). In some assays, the tetrapeptide Gly-Pro-Arg-Pro was added to 3.5 % NaCl. In addition, CaCl 2 was also substituted with 3.5 % NaCl, to prevent clot formation. All components were used at room temperature. The killing activity of serum and plasma was determined by counting the number of dead ciliates per well at several different times. In all experiments triplicate samples were analysed, and the experiment was repeated three times.
Plasma and serum were heated to 45 o C for 30 minutes. Heating the plasma and serum inactivates complement and also the coagulation capacity of plasma. In some experiments, we used pooled plasma and serum from 5 immunized fish. The experiment was carried out under aseptic conditions. Ten µl of ciliates (250 ciliates in 3.5 % NaCl), 9 µl of several dilutions of pooled fresh and heated serum or plasma in 3.5% NaCl, 5 µl of 3.5 % NaCl, and 15 µl of 40 mM CaCl 2 in distilled water were added to each well (total 39 µl). In some assays, the tetrapeptide Gly-Pro-Arg-Pro was added to 3.5 % NaCl. In addition, CaCl 2 was also substituted with 3.5 % NaCl, to prevent clot formation. All components were used at room temperature. The killing activity of serum and plasma was determined by counting the number of dead ciliates per well at several different times. In all experiments triplicate samples were analysed, and the experiment was repeated three times.
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