PrimeSTAR HS DNA Polymerase was purchased from TaKaRa; Restriction endonucleases and T4 DNA ligase were obtained from Fermentas; and the nucleotides NADPH, NADH, NAD+, and NADP+ were purchased from Roche and prepared just before use, ATP, ADP, AMP, diamide, glucose, oxamate, dehydroepiandrosterone, carmustine, dorsomorphin, malate, isocitrate, 2-deoxy-d-glucose, MTT, glucose-6-phosphate, 6-phosphogluconate, phenazine methosulfate, yeast G6PD, NAM, thiazolylblue, phenazine ethosulfate and lipopolysaccharides (LPS) were all obtained from Sigma-Aldrich. Glutamine and pyruvate were provided from Invitrogen. Digitonin and H2O2 were obtained from Calbiochem (Merck, Germany), and KP372-1 was purchased from Echelon Biosciences, Inc. Recombinant murine interferon-γ (IFN-γ) was obtained from Absin. Other reagents were of analytical grade and obtained from local suppliers.
Adenosine diphosphate (adp)
Manufactured by Roche
Sourced in France, Switzerland, Germany
The ADP is a laboratory instrument that measures the concentration of adenosine diphosphate (ADP) in a sample. ADP is a key molecule involved in cellular energy metabolism. The ADP functions as a core measurement tool to quantify ADP levels, providing important data for research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Adenosine triphosphate (atp), by Roche (4 mentions)
Multiplate analyzer, by Roche (4 mentions)
Arachidonic acid, by Roche (2 mentions)
Mili q water, by Merck Group (2 mentions)
Mant adp, by Thermo Fisher Scientific (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Mant atp, by Thermo Fisher Scientific (2 mentions)
Mant dadp, by Jena Biosciences (2 mentions)
Mant datp, by Jena Biosciences (2 mentions)
Mantatp, by Jena Biosciences (2 mentions)
Amp pnp, by Roche (2 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
2 deoxy d glucose, by Merck Group (1 mentions)
T4 dna ligase, by Thermo Fisher Scientific (1 mentions)
Restriction endonuclease, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Glucose 6 phosphate (g6p), by Merck Group (1 mentions)
Pyruvate, by Thermo Fisher Scientific (1 mentions)
20 protocols using adenosine diphosphate (adp)
1
Detailed Reagent Acquisition Protocol
2
Platelet Aggregation Assay Reagents
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ADP was obtained from Roche (Boulogne-Billancourt, France) and PAR1ap (activating peptide of protease-activated receptor 1), used as a thrombin receptor activator, from Bachem (Bubendorf, Switzerland). Collagen was purchased from Nycomed (kit SKF Solution, Austria Takeda Pharmaceuticals International, Zurich, Switzerland).
PJ34 was purchased from Sigma-Aldrich (Saint Quentin Fallavier, France), DPQ from Santa Cruz Biotechnology (CliniSciences, Nanterre, France) and INO-1001 from Seleck Chemicals (Euromedex, Souffelweyersheim, France).
All reagents were dissolved and diluted in distilled water, except DPQ that was dissolved in 100% DMSO and diluted in distilled water.
PJ34 was purchased from Sigma-Aldrich (Saint Quentin Fallavier, France), DPQ from Santa Cruz Biotechnology (CliniSciences, Nanterre, France) and INO-1001 from Seleck Chemicals (Euromedex, Souffelweyersheim, France).
All reagents were dissolved and diluted in distilled water, except DPQ that was dissolved in 100% DMSO and diluted in distilled water.
3
Measuring Glutamate Dehydrogenase Activity
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To measure PAG activity, 41 U GDH (Serva Electrophoresis, GmbH; Heidelberg, Germany), dissolved in 50% glycerol, was placed on glass slides and equally distributed by spreading the solution across the glass slide with a coverglass. The glass slides were dried at room temperature in a vacuum (<1-6 bar). Afterwards, 7-µm-thick cryostat tissue sections were cut (for lung tissues, 8-µm-thick sections were cut), captured onto GDH-coated glass slides, and stored at -80C. Before incubation, tissue sections were air dried at room temperature for 30 min.
In order to ensure excess GDH activity and its even spreading over the glass, GDH activity was measured by incubating the GDH films after pouring on top of the films incubation medium containing 18% PVA (Sigma-Aldrich; St. Louis, MO), 0.1 M phosphate buffer (pH 8.0), 5 mM NitroBT (Sigma-Aldrich), 3 mM NAD+ (Roche Applied Science; Basel, Switzerland), 2 mM ADP (Roche Applied Science), and 0.32 mM PMS (Serva Electrophoresis, GmbH) in the presence of 10 mM glutamate (Merck; Darmstadt, Germany). NitroBT was dissolved by heating NitroBT in a 1:1 solution of 100% ethanol and dimethylformamide with a final concentration of 2% v/v (0.34 M ethanol and 0.26 M dimethylformamide) for each solvent.
In order to ensure excess GDH activity and its even spreading over the glass, GDH activity was measured by incubating the GDH films after pouring on top of the films incubation medium containing 18% PVA (Sigma-Aldrich; St. Louis, MO), 0.1 M phosphate buffer (pH 8.0), 5 mM NitroBT (Sigma-Aldrich), 3 mM NAD+ (Roche Applied Science; Basel, Switzerland), 2 mM ADP (Roche Applied Science), and 0.32 mM PMS (Serva Electrophoresis, GmbH) in the presence of 10 mM glutamate (Merck; Darmstadt, Germany). NitroBT was dissolved by heating NitroBT in a 1:1 solution of 100% ethanol and dimethylformamide with a final concentration of 2% v/v (0.34 M ethanol and 0.26 M dimethylformamide) for each solvent.
4
ATPase activity assay of p53 variants
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NADH coupled microtiter plate assay was performed as described in published protocol [20] (link). ATPase assay time-course was performed with varying proteins concentrations (as indicated in figure legends) up to 180 minutes at 30°C in ATPase buffer (described in [20] (link)) with 1 mM NADH. NADH absorbance decline was measured at 340 nm in 96 well plate using Tecan Infinite M-200 spectrophotometer. ATPase buffer in the absence of proteins was taken as the negative control. ATPase buffer containing 5 mM ADP (Roche) was taken as positive control to verify the activity of coupled reaction.
Radioactive ATPase assay was performed using thin layer chromatography (TLC) with 2 μM each of GST tagged full length wildtype p53 (p53-FL), p53-3C, p53-24, p53-25 or p53-35 incubated with 0.25 μM of 50 nCi/μl [α-32P] ATP (BRIT, Hyderabad, India) in 25 mM Tris–HCl (pH 7.6), 1.8 mM DTT and 13 mM magnesium acetate at 30°C for 30 minutes. The reactions were stopped with 0.2% SDS, heated at 100°C for 3 minutes and spotted on polyethyleneimine sheet (Merck) that was developed in 0.75 M KH2PO4 (pH 3.6). The dried PEI sheet was scanned using Molecular Dynamics Storm 820 PhosphorImager. The percentage of [α-32P] ATP hydrolyzed to [α-32P] ADP was quantified by Image J software.
Radioactive ATPase assay was performed using thin layer chromatography (TLC) with 2 μM each of GST tagged full length wildtype p53 (p53-FL), p53-3C, p53-24, p53-25 or p53-35 incubated with 0.25 μM of 50 nCi/μl [α-32P] ATP (BRIT, Hyderabad, India) in 25 mM Tris–HCl (pH 7.6), 1.8 mM DTT and 13 mM magnesium acetate at 30°C for 30 minutes. The reactions were stopped with 0.2% SDS, heated at 100°C for 3 minutes and spotted on polyethyleneimine sheet (Merck) that was developed in 0.75 M KH2PO4 (pH 3.6). The dried PEI sheet was scanned using Molecular Dynamics Storm 820 PhosphorImager. The percentage of [α-32P] ATP hydrolyzed to [α-32P] ADP was quantified by Image J software.
5
Eosinophil-Mediated Modulation of Plasma Clotting
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Human standard citrate plasma (Siemens Healthcare) was freed from residual cellular and subcellular particles by ultracentrifugation and supplemented with 10% rat plasma (GeneTex). Plasma clotting time with or without addition of eosinophils was automatically recorded after recalcification with 20 µl of star-tem (ROTEM; Tem International GmbH) using a ball coagulometer (BC1; SYCOmed) at 37°C. When indicated, eosinophils were stimulated with 40 µM ADP (Roche) or 20 µg/ml collagen (Roche) for 10 min at 37°C before recalcification. Clotting index was calculated as follows: clotting time [sec.]−1 × 100.
6
Multiplate Impedance Aggregometry for Platelet Function
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Impedance aggregometry was performed with the Multiplate analyzer (Roche Diagnostics, Mannheim, Germany) as previously described [10 (link)]. After addition of 6.4 μM ADP (Roche Diagnostics, Mannheim, Germany) to hirudin-anticoagulated blood, the adhesion of activated platelets to the electrodes led to an increase in impedance, which was detected for each sensor unit separately and transformed to aggregation units (AU) that were plotted against time. One AU corresponds to 10 AU ∗ min (area under the curve of AU). Multiple electrode aggregometry (MEA) in response to ADP was available for 293 patients (97.3%) of group 1 and for all patients (100%) of group 2.
7
Synthesis and Analysis of Inositol Phosphates
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Prostaglandin E1 was obtained from Cayman Chem. Comp. (Ann Arbor, MI, USA); BCA-protein-assay-reagent from Pierce (Rockfield, IL, USA); pentobarbital-sodium (Nembutal R) from Wirtschaftsgenossenschaft dtsch. Tierärzte (Hannover, Germany); ATP, GTP, UTP, CTP, ADP, and GDP were obtained from Roche Diagnostics (Mannheim, Germany); inositol(1,4)P2, inositol(2,4)P2, inositol(4,5)P2, inositol(5,6)P2, inositol(1,4,5)P3, noradrenaline, adrenaline, and silicic acid SIL-350 were purchased from Millipore-Sigma (Darmstadt, Germany); Sephadex G15, HL-Q-Sepharose HP, [5,6(n)-3H] PGE1, [8-3H] GTP, [8-14C] ADP, [2-3H] inositol with stabilizer PT6-271, [2-3H] inositol(1,4)P2, and [3H] inositol(1,4,5)P3 were obtained from GE-Healthcare (Solingen, Germany); activated charcoal, toluene, ethyl acetate, methanol, Rotiscint 11, and Rotiscint 22 were obtained from Carl Roth (Karlsruhe, Germany); all other chemicals of reagent grade were from E. Merck (Darmstadt, Germany).
A regiospecific ionsitol-P5/inositol-P4-phosphohydrolase from Dictyostelium discoideum was exploited for the synthesis of inositol (1,6)-bisphosphate [35 (link)].
Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 8 were drawn with Microsoft office Excel 2007 (12.0.6787.5000) SP3 MSO (12.0.6785.5000).
A regiospecific ionsitol-P5/inositol-P4-phosphohydrolase from Dictyostelium discoideum was exploited for the synthesis of inositol (1,6)-bisphosphate [35 (link)].
8
Multiplate Platelet Aggregometry Assay
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Hirudin‐anticoagulated blood was analyzed using multiple electrode impedance platelet aggregometry (Multiplate, Diapharma, West Chester, Ohio) 30 minutes after blood collection according to the manufacturer's instructions. Briefly, 300 μL of anticoagulated blood was incubated with 300 μL of 0.9% NaCl for 3 minutes at 37°C with continuous stirring, followed by the addition of 6.5 μM ADP (Roche, Basel, Switzerland) or 0.5 mM arachidonic acid (AA) (Roche, Basel, Switzerland). Each test cell incorporates a duplicate sensor for 2 simultaneous measurements, which served as an internal control. Platelet aggregation was reported as area under the curve over a 6‐minute interval. If the Pearson's correlation coefficient and difference between the 2 measurements were <0.98 or >20%, respectively, the measurements were repeated.
9
Characterization of Nucleotide Binding
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All chemical reagents were of the highest purity commercially available. Millipore MiliQ® water (filtered through a 0.2-μm filter) or Sigma RNase-free water was used in all buffers. ATP (Sigma A7699, ≥ 99% purity assayed by HPLC, ≤ 0.1% inorganic phosphate) and ADP (Roche Molecular Biochemicals) concentrations were determined by absorbance using ε259 of 15,400 M− 1 cm− 1. Polyuridylic acid (polyU RNA) (SC-215733A; Santa Cruz Biotechnology) was dialyzed extensively against distilled deionized water and ethanol precipitated prior to use. RNA concentrations were determined by absorbance using ε260 of 9660 M− 1 cm− 1 and are in units of nucleotides. Mant-labeled nucleotides (2′ and 3′ mixed isomer, mantATP, and mantADP from Invitrogen;mantATP, mant-dATP, and mant-dADP from Jena Biosciences) concentrations were determined by absorbance (ε255 = 23,300 M− 1 cm− 1). One molar equivalent of MgCl2 was added to all nucleotides immediately before use.
All assays were performed at 25 ± 0.1 °C in 30 mM Hepes(pH 7.5), 100 mM KCl, 2 mM MgCl2, and 2 mM DTT.
All assays were performed at 25 ± 0.1 °C in 30 mM Hepes(pH 7.5), 100 mM KCl, 2 mM MgCl2, and 2 mM DTT.
10
Characterization of Nucleotide Interactions
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