To measure signs of intracerebral bleeding, haemoglobin levels in the ischaemic hemisphere of freshly extracted and homogenised brains were determined by a spectrophotometric assay using Drabkin’s reagent (Sigma-Aldrich).[35 (link)] A standard curve was obtained with known amounts of haemoglobin (Sigma-Aldrich).
Haemoglobin
Manufactured by Merck Group
Sourced in United States, Poland
Haemoglobin is a protein found in red blood cells that carries oxygen from the lungs to the body's tissues and carbon dioxide from the tissues back to the lungs. It is a key component in the analysis of blood samples.
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Lab products found in correlation
Linoleic acid, by Merck Group (2 mentions)
Bile extract, by Merck Group (2 mentions)
Ammonium thiocyanate, by Merck Group (2 mentions)
Pepsin, by Merck Group (2 mentions)
α amylase, by Merck Group (2 mentions)
Pancreatin, by Merck Group (2 mentions)
Ferrozine, by Merck Group (2 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (2 mentions)
Lysozyme, by Merck Group (2 mentions)
Drabkin s reagent, by Merck Group (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Plastic coverslip, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Collagenase, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Glutathione reductase, by Merck Group (1 mentions)
10 protocols using haemoglobin
1
Quantifying Intracerebral Hemoglobin Levels
2
Colorimetric Enzyme Assay on Paper
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Hydrogen peroxide, the co-substrate of MPO, was generated in the system by the conversion of glucose with glucose oxidase (GOX) from Aspergillus niger. The GOX solution (2 mg/ml) was applied by drop casting (2.5 l) on the centre of the paper strip before the immobilised substrate mixture in the sample flow direction, and dried with air stream. The oxidation of the immobilised substrate mixture to its coloured product was performed using purified MPO. MPO was diluted in sodium phosphate buffer (200 mM, pH 6.5) to different final concentrations . The MPO solution (60l) was applied at the tip of the paper strip close to the immobilised GOX inducing a liquid flow that first meets and drags GOX and finally encounters the immobilised substrates. When haemoglobin interference was assessed, haemoglobin (Sigma-Aldrich ® ) was diluted in sodium phosphate buffer (200 mM, pH 6.5) to final concentrations of 0.01 and 0.001 mM and applied to the paper strip in the same manner as the MPO. In all the cases once the flow passed through the entire length of the strip the incubation time (5 min) at RT was initiated. After the incubation period the colour change was measured using a spectroflash ® SF300 with CIE Standard Illuminat D65 according to the CIELab concept.
As a reference, a paper strip with immobilized substrate, but without enzyme was used.
As a reference, a paper strip with immobilized substrate, but without enzyme was used.
3
Quantification of Metabolic Parameters
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Kits for glucose, insulin, total cholesterol, triglycerides, HDLc and LDLc were purchased from Abbott Laboratories (Abbott Park, IL, USA). HsCRP kits were supplied by Beckman Corp (Brea, A, USA). The HbA1c kit was purchased from Menarini Diagnostics (Florence, Italy). The MPO kit was purchased from Cayman Chemical (Michigan, USA).
Glucose, trypan blue, arginine, glutathione reductase, H2O2, haemoglobin, RPMI1640 supplemented with 20 mM HEPES, HBSS, TNF-α, human serum albumin (HSA, Albuminate 25%) and fibronectin were obtained from Sigma-Aldrich (Sigma Chem. Co., St. Louis, MO, USA). Dextran was acquired from Fluka (St. Louis, MO, USA). HBSS was supplied by Cambrex (Verviers, Belgium). DCFH-DA and Mitosox tracker were provided by Calbiochem (San Diego, CA, USA). Dulbecco’s PBS—with (DPBS+) or without (DPBS-) Ca2+ and Mg2+—endothelial cell growth medium culture media and fetal bovine serum were obtained from LONZA (Verviers, Belgium). Plastic coverslips (diameter of 25 mm) were purchased from Nunc (Thermo Fisher Scientific). PBS, collagenase, and trypsin-EDTA were obtained from Invitrogen (Eugene, OR, USA). Ficoll-Paque TM Plus was purchased from GE Healthcare (Little Chalfont, Buckinghamshire,UK).
Glucose, trypan blue, arginine, glutathione reductase, H2O2, haemoglobin, RPMI1640 supplemented with 20 mM HEPES, HBSS, TNF-α, human serum albumin (HSA, Albuminate 25%) and fibronectin were obtained from Sigma-Aldrich (Sigma Chem. Co., St. Louis, MO, USA). Dextran was acquired from Fluka (St. Louis, MO, USA). HBSS was supplied by Cambrex (Verviers, Belgium). DCFH-DA and Mitosox tracker were provided by Calbiochem (San Diego, CA, USA). Dulbecco’s PBS—with (DPBS+) or without (DPBS-) Ca2+ and Mg2+—endothelial cell growth medium culture media and fetal bovine serum were obtained from LONZA (Verviers, Belgium). Plastic coverslips (diameter of 25 mm) were purchased from Nunc (Thermo Fisher Scientific). PBS, collagenase, and trypsin-EDTA were obtained from Invitrogen (Eugene, OR, USA). Ficoll-Paque TM Plus was purchased from GE Healthcare (Little Chalfont, Buckinghamshire,UK).
4
Growth Conditions for Group B Streptococcus
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GBS strains (S1 Table ) were routinely grown in Brain Heart Infusion (BHI) broth overnight under static conditions in a 5% CO2/95% air incubator at 37°C to prepare pre-cultures. Growth experiments were carried out using BHI, Todd Hewitt Broth (THB) or M17 broth with riboflavin 5 μM (M17rib) and supplemented with different sugars (0.5–1% glucose and/or 0.5% galactose, or 0.5% lactose). Cultures were usually inoculated at OD600 = 0.025 in M17rib and incubated under static conditions. For aeration conditions, M17 broth was supplemented with 50 μg ml-1 of haemoglobin (Sigma). Culture volume was 1/10 of flask volume, with agitation at 125–200 rpm. For respiration, aerated cultures were supplemented with 30 μM of menaquinone (MK-4 Sigma). Escherichia coli strain TG1 was used as host strain for cloning. When required, antibiotics were used as follows: for GBS, erythromycin (Ery) 1 μg.ml-1, chloramphenicol (Cm) 5 μg.ml-1, and for E. coli, Ery 150 μg.ml-1, ampicillin (Amp) 100 μg.ml-1. For electroporation, GBS strains were grown in BHI.
5
Evaluating Antioxidant and Enzymatic Properties
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Ferrozine (3-(2-pyridyl)-5,6-bis-(4-phenyl-sulfonic acid)-1,2,4-triazine), ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) α-amylase, pancreatin, pepsin, bile extract, Folin-Ciocalteau reagent, linoleic acid, ammonium thiocyanate, and haemoglobin were purchased from Sigma-Aldrich Company (Poznan, Poland). All others chemicals were of analytical grade.
6
Protein Screening with Molecularly Imprinted Polymers
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Six proteins were tested. Model proteins were utilized for automated screening trials with MIPs to benchmark the technique. These included lysozyme (hen egg white; Sigma, catalogue No. L7651), thaumatin (from Thaumatococcus daniellii; Sigma, catalogue No. T7638), trypsin (bovine pancreatic; Sigma, catalogue No. T4665) and haemoglobin (bovine; Sigma, catalogue No. H2500). The model proteins were prepared at the required concentrations in deionized water. Two target proteins, Chlamydia trachomatis plasmid-encoded immunodominant antigen (Pgp3; 7 mg ml−1 in 220 mM sodium chloride, 50 mM Tris–HCl pH 7.5 with 5 mM DTT) and human macrophage migration inhibitory factor (MIF; 11 mg ml−1 in 20 mM Tris pH 7.5, 20 mM sodium chloride), were utilized for the automated optimization trials with MIPs.
7
Fasting Animal Feeding Protocol
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Unfed animals (25–30 days fasting) were fed the following meals using an artificial feeding apparatus at 37°C: rabbit blood, plasma (obtained from heparinized rabbit blood) or Tyrode's physiological solution (0.14 M NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.4 mM NaH2PO4, 0.1% glucose) containing 100 μM ATP. Tyrode's solution was supplemented with 20 μM hemin, 50 mg/ml haemoglobin (Sigma), 50 mg/ml bovine albumin (Sigma), 50 μM MitoTEMPO (Sigma) or an amino acids mixture as described in Hara et al. [15 (link)] (L -Arg, 84 mg/l; L -Cys, 48 mg/l; L -Glu, 584 mg/l; L -His, 42 mg/l; L -Ile, 105 mg/l; L -Leu, 105 mg/l; L -Lys, 146 mg/l; L -Met, 30 mg/l; L -Phe, 66 mg/l; L -Thr, 95 mg/l; L -Trp, 16 mg/l; L -Tyr, 72 mg/l; L -Val, 94 mg/l; L -Gln, 103.75 mg/l) [15 (link)].
8
Purification of Voltage-Dependent Anion Channel
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Phase partitioning was conducted according to published methods [50 (link)]. Control proteins haemoglobin and lysozyme were purchased from Sigma–Aldrich. Arabidopsis thaliana voltage-dependent anion channel 1 (AtVDAC1) was refolded and purified according to published methods [51 (link)]. All temperature incubations were for a period of 3 min. Protein solutions were made up to 1 mg/ml in 10 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% Triton X-114 at 0°C. A 200 μl of sucrose cushion was prepared consisting of 6% (w/v) sucrose, 10 mM Tris/HCl, pH 7.4, 150 mM NaCl and 0.06% Triton X-114. Fifty microlitres of protein solution was overlaid on the sucrose cushion and incubated at 30°C before centrifugation at 300 g for 3 min. The upper aqueous layer (70 μl) was removed and additional Triton X-114 was added to 0.5 % concentration. After incubation at 0°C and gentle mixing, this aqueous phase was overlaid on the same sucrose cushion, incubated at 30°C and then centrifuged at 300 g for 3 min to pellet the detergent phase. The upper aqueous phase was removed and received an additional 2% Triton X-114. This was then cooled to 0°C, mixed, then incubated at 30°C before a final centrifugation at 300 g for 3 min (pellet discarded). The supernatant (aqueous phase) was then analysed by SDS/PAGE alongside the previous detergent phase.
9
Haemoglobin Degradation Compounds Analysis
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Iron-containing compounds in the degradation process of haemoglobin were purchased for MPI scanning: haemoglobin (H7379; Sigma–Aldrich, USA), ferrous-stabilised haemoglobin (H0267; Sigma–Aldrich, USA) and ferritin protein (ab96514; Abcam, USA).
10
Antioxidant and Enzymatic Assays
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Ferrozine (3-(2-pyridyl)-5,6-bis-(4-phenyl-sulfonic acid)-1,2,4-triazine), ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)), NBT (nitro blue tetrazolium), DETAPAC (diethylenetriaminepentaacetic acid), α-amylase, pancreatin, pepsin, bile extract, Folin-Ciocalteu reagent, linoleic acid, ammonium thiocyanate, and haemoglobin were purchased from Sigma-Aldrich company (Poznan, Poland). All other chemicals were of analytical grade.
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