GSAO and control compound 4‐(N‐(S‐glutathionylacetyl)amino)benzoic acid were synthesized previously as described25 and conjugated to amine‐reactive succinimidyl ester Alexa Fluor 647‐NHS (Life Technologies). Confirmation of GSAO activity and batch to batch validation testing after conjugation were performed as previously described.17 , 26 Monomeric collagen‐related peptide (CRP, Auspep) was cross‐linked using N‐succinimidyl 3‐(2‐pyridyldithio)propionate (Sigma‐Aldrich) to produce CRP‐xL. Reagents are available on request.
C reactive protein (crp)
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Spain
The CRP is a laboratory equipment used for the quantitative determination of C-Reactive Protein (CRP) in human serum or plasma. CRP is a general marker of inflammation and infection in the human body.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 6 (il 6), by Merck Group (3 mentions)
96 well microplate, by SPL Life Sciences (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Nt probnp, by Abcam (2 mentions)
Tnf α, by Abcam (2 mentions)
Svcam1, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dextran sulfate sodium (dss), by Merck Group (1 mentions)
Tmb substrate kit, by Thermo Fisher Scientific (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
3 3 5 5 tetramethylbenzidine tmb substrate kit, by Thermo Fisher Scientific (1 mentions)
Xhoi, by New England Biolabs (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Fibrinogen, by Abcam (1 mentions)
Desmosine, by MyBioSource (1 mentions)
25 protocols using c reactive protein (crp)
1
Fluorescent Labeling of GSAO and CRP
2
Immunoassay Protocol for HRP and CRP
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GA, DSS, DSG, BS(PEG)5, HRP, CRP, tween-20, Triton X-100, isopropyl β-D-1-thiogalactopyranoside (IPTG), ethylenediaminetetraacetic acid (EDTA), glycine and hydrochloric acid were purchased from Merck (Darmstadt, Germany). Rabbit polyclonal antibodies against HRP, rabbit polyclonal antibodies against CRP and fluorescein-conjugated goat polyclonal antibodies against CRP were obtained from Abcam (Cambridge, UK). Phosphate-buffered saline (PBS) was purchased from LPS solution (Daejeon, Korea) and used as an antibody-dissolving buffer. In addition, 96-well microplates were obtained from SPL Life Sciences (Pocheon, Korea). The TMB substrate kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
3
Immunoassay Reagents and Materials
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Goat polyclonal antibodies against C-reactive protein (CRP), goat polyclonal antibodies against horseradish peroxidase (HRP), goat polyclonal antibodies against CRP labeled with HRP, and fluorescein-conjugated rabbit polyclonal antibodies against human immunoglobulin (Ig) G were obtained from Abcam (Cambridge, UK). Tris-HCl buffer ( pH 8.0), Tris-glycine buffer, phosphate-buffered saline (PBS), Luria-Bertani (LB) broth, ampicillin, protein G, and sodium dodecyl sulfate (SDS)-loading buffer were purchased from LPS solution (Daejeon, Korea). 96-Well microplates were obtained from SPL Life Sciences (Pocheon, Korea). The 3,3′,5,5′-tetramethylbenzidine (TMB) substrate kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Triton X-100, isopropyl β-D-1-thiogalactopyranoside (IPTG), sucrose, lysozyme, HRP, CRP, and other analytical-grade chemicals were obtained from Merck (Darmstadt, Germany). The restriction enzymes, XhoI and KpnI, and ligase were purchased from New England Biolabs (Ipswich, MA, US).
4
Measurement of Inflammatory Biomarkers
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Plasma levels of biomarkers were measured using commercially available ELISA kits: C-reactive protein (CRP) (Sigma, St Louis, MO, USA), desmosine (MyBiosource, San Diego, CA, USA), 8-isoprostane, prostaglandin E2, thromboxane B2, resolvin D1, resolvin D2, resolvin E1, leukotriene E4 (Cayman, Ann Arbor, MI, USA), fibrinogen (Abcam, Cambridge, MA, USA), intracellular adhesion molecule (ICAM)-1 (R&D Systems), 4-hydroxynonenal, malondialdehyde (Cell Biolabs, San Diego, CA, USA) and lipoxin A4 (TSZ ELISA, Waltham, MA, USA). Urine samples were analysed for 8-isoprostane, leukotriene E4, desmosine and 8-oxo-dG (HT 8-Oxo-dG Human ELISA; Trevigen, Gaithersburg, MD, USA). Salivary biomarkers IL-1β, IL-6, prostaglandin E2, resolvin D1 and resolvin D2 were also measured. EBC samples were analysed for 8-isoprostane. All these biomarkers were measured quantitatively as per the manufacturers' instructions.
5
Influenza A Hemagglutinin Protein Analysis
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The influenza A H1N1 (A/New Caledonia/20/1999) Hemagglutinin/HA Protein (His Tag) and influenza A H5N1 (A/VietNam/1203/2004) Hemagglutinin/HA Protein (His Tag and fragment crystallizable (Fc) Tag) were purchased from Sino Biological (Beijing, China). Cysteamine, myoglobin, hemoglobin, bovine serum albumin (BSA), potassium hexacyanoferrate(III) and potassium hexacyanoferrate(II) trihydrate ([Fe(CN)6]3−/4−), and C-reactive protein (CRP) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Silver nitrate (AgNO3) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Daejung Chemical and Metals Co., Ltd. (Siheung, South Korea). For the clinical test, the human serum from human male AB plasma (USA origin) was purchased from Sigma–Aldrich (St. Louis, MO, USA). The four fragments of DNA 4WJ were synthesized by Bioneer (Daejeon, South Korea). All oligonucleotides were supplied by Bioneer and diluted in nuclease-free water. Table 1 showed the DNA sequences were as follows:
6
Biosensing Platform for NT-proBNP and Galectin-3
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Tetrachloroauric (III) acid trihydrate (HAuCl4·3H2O, ≥99.9%), sodium citrate tribasic dihydrate (Na3C6H5O7·2H2O, ≥99.0%), phosphate-buffered saline (PBS) pH 7.4, trizma hydrochloride (C4H11NO3·HCl, 99%), bovine serum albumin (BSA) from bovine (≥98%), C-reactive protein (CRP) from human fluids (≥90%), urea (CH4ON2, 99%), α-amylase from porcine pancreas (≥10 units/mg) and fetal bovine serum were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethylenediaminetetraacetic acid (C10H16N2O8, 99%) was purchased from PanReac AppliChem. The aptamer specific for NT-proBNP was a single-stranded (ss) DNA with the following sequence (5′-GGCAGGAAGACAAACAGGTCGTAGTGAAACTGTCCACCGTAGACCGGTTATCTGTTGGTCTGTGGTGCTGT-3′, MW: 22,383.6 g·mol−1, ε: 705,500 L·mol−1·cm−1) purchased from Eurogentec. The NT-proBNP (HPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPRSPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH, >95%) was acquired from abcam. The galectin-3 (98%) was purchased from RayBiotech. The reagents were used as acquired. Ultra-pure water was obtained using the Milli-Q system using a 0.22 µm filter (Synergy equipment, Millipore (Burlington, MA, USA)).
7
Comprehensive Rat Blood Analysis
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Three milliliters of whole blood were collected from each rat into potassium-EDTA tubes for immediate measurement of the white blood cell count using automated Cobas 6800/8800 systems (Roche Group, Switzerland). Additional blood was collected into plain tubes and centrifuged at 3000×g and 5 °C for 15 min. The obtained serum was stored at − 80 °C until use. One milliliter of serum per rat was used for lipid profiling (TC, TG, LDL-cholesterol, and HDL-C), the assessment of renal function (blood urea nitrogen (BUN) and creatinine concentrations), and liver enzyme activity measurement (alanine aminotransaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ASP)) using the Cobas 6800/8800 systems. Two hundred microliters of serum were used to measure the concentrations of tumor necrosis factor-alpha (TNF-α) (Abcam: Cambridge, UK) and c-reactive protein (CRP) (Sigma-Aldrich, Inc.: St. Louis, MO, USA) using rat elisas, according to the manufacturers’ instructions.
8
Nanoimprinted Optofluidic Biosensor Protocol
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Sodium phosphate buffer (PBS, 8 mM Na 2 HPO 4 , 2 mM, 137 mM NaCl, 2.7 mM KCl, pH 7.4), PBS-T (PBS with polysorbate 20 0.05% v/v), were prepared with purified water (Milli-Q, Millipore Iberica, Darmstadt, Germany) and filtered through 0.2 μm polyethersulfone membranes (Merck, Darmstadt, Germany). Polydimethylsiloxane (PDMS) Sylgard 184 was from Dow Corning (Wiesbaden, Germany). Bovine serum albumin (BSA), polysorbate 20 (Tween 20), antiBSA rabbit IgG, C-reactive protein (CRP), casein and human serum (human male, AB plasma) were supplied by Sigma-Aldrich (Madrid, Spain). Single-mode optical fibers SMF-28 were purchased from Corning (Madrid, Spain). The silicon grooved nanostructure (555.5 nm period, 140 nm groove depth, duty cycle 50%) used as a master to prepare the micro-contact printing stamp, was from LightSmyth (Eugene, OR, USA).
9
Assessing Blood Markers for Thermal Exposure
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Blood samples were taken from an antecubital vein from the participant's nondominant arm, using a 20ml syringe and 21G hypodermic needle, while participants were sitting upright and resting. To assess the impact of longer term changes to the variables we measured, blood samples were taken pre and post the 7wk no-heat exposure period. For determining the acute responses blood was taken immediately before and as soon as possible after Wear 1 and Wear 2 of the 4wk training course.
Plasma was obtained by centrifugation (2500g for 15 min in a refrigerated centrifuge)
and stored frozen (-85C) for analysis when all samples had been collected.
Whole blood samples were assessed for blood cell count using automated flow cytometry with electrical impedance and light detectors (full blood count analyser, Sysmex, Europe). Values for white blood cells (WBC) and white blood cell content (neutrophils, eosinophils, basophils, lymphocytes and monocytes) were analysed.
While haemoglobin (Hb) and haematocrit (Hct) estimated change in plasma volume (∆PV) (Dill and Costill, 1974) .
Plasma was thawed before being analysed for C-reactive protein (C-RP) (Sigma Aldrich, UK), cortisol (Enzo Life Sciences, UK), IL-6 (Sigma Aldrich, UK) and Immunoglobulin G (IgG) (Enzo Life Sciences, UK) using ELISA.
Plasma was obtained by centrifugation (2500g for 15 min in a refrigerated centrifuge)
and stored frozen (-85C) for analysis when all samples had been collected.
Whole blood samples were assessed for blood cell count using automated flow cytometry with electrical impedance and light detectors (full blood count analyser, Sysmex, Europe). Values for white blood cells (WBC) and white blood cell content (neutrophils, eosinophils, basophils, lymphocytes and monocytes) were analysed.
While haemoglobin (Hb) and haematocrit (Hct) estimated change in plasma volume (∆PV) (Dill and Costill, 1974) .
Plasma was thawed before being analysed for C-reactive protein (C-RP) (Sigma Aldrich, UK), cortisol (Enzo Life Sciences, UK), IL-6 (Sigma Aldrich, UK) and Immunoglobulin G (IgG) (Enzo Life Sciences, UK) using ELISA.
10
Purification and Labeling of Immune Proteins
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Human Clu was purified from plasma, as previously described.7 (link) Recombinant human MBL and human and murine Clu were from Biotechne (Lille, France). HSA, C1q, and FITC-labeled WGA were from Sigma-Aldrich (St. Louis, MO, USA). SAP (Calbiochem, Darmstadt, Germany), CRP (Millipore, Billerica, MA, USA), and H1, H2A, H2B, H3, and H4 histone subunits (New England Biolabs, Ipswich, MA, USA) were from the indicated providers. Proteins were labeled with Oregon green 488 dye (FluoReporter Oregon Green 488 Protein Labeling Kit; Invitrogen Molecular Probes, Carlsbad, CA, USA) or biotinylated (EZ-Link Sulfo-NHS-LC-Biotin Kit; Pierce, Rockford, IL, USA) using the commercial kits. The origins and clone numbers of the mAbs used in this study are listed in Table 1 .
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