Serum total protein concentration and the analytical curve for serum albumin were determined. Both the examined samples and the reference samples were run in parallel in three repetitions. Sample absorbance was measured using Multiskan Ascent Microplate Photometer (Thermo Labsystems, Philadelphia, PA, USA) at λ = 562 nm and total protein concentration was calculated from the standard curve equation. The serum concentration of PON1, PON2, PON3 and MPO proteins was determined using the Human PON1, PON2, PON3 and MPO ELISA Kit (MyBiosource San Diego CA, USA) according to the protocols provided by the manufacturer. β-actin was used for endogenous control of protein concentration in the samples and determined with the help of the Human Actin Beta (ACTb) ELISA Kit (BMASSAY) based on the manufacturer’s recommendations. The absorbance of the samples was measured using a Multiskan Ascent Microplate Photometer (Thermo Labsystems, Philadelphia, PA, USA) at λ = 450 nm. Analytical curves were constructed for the analyzed proteins to determine the protein concentration.
Multiskan ascent microplate photometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Multiskan Ascent Microplate Photometer is a laboratory instrument designed for absorbance measurements in microplates. It is capable of performing absorbance readings at various wavelengths to support a range of assays and applications.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Micro bcatm protein assay kit, by Thermo Fisher Scientific (1 mentions)
Wizard2, by PerkinElmer (1 mentions)
Human il 23 quantikine elisa kit, by R&D Systems (1 mentions)
Human foxp3 elisa kit, by MyBioSource (1 mentions)
Human il 17 quantikine elisa kit, by R&D Systems (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
13 protocols using multiskan ascent microplate photometer
1
Serum Protein Profiling for Oxidative Stress
2
Protein Concentration Determination in Serum
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150 µL of the reaction mixture was added to pits containing 150 µL of serum, diluted 10 times in 10 mM of phosphate buffered saline, pH 7.4, and incubated (2 h, 37 °C). To specify protein concentration, an analytical curve for serum albumin was determined. Both the examined samples and the reference samples were made parallel in three repetitions. Sample absorbance was measured using Multiskan Ascent Microplate Photometer (Thermo Labsystems, Philadelphia, PA, USA) at λ = 562 nm and total protein concentration was calculated from the standard curve equation.
3
Serum Cytokine Profiling Using ELISA
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The concentration of proteins IL-1, IL-6 and IL-10 in the patients’ serum was determined using specific Elisa kits (R&D Systems, Inc, Minneapolis, MN, USA) for each cytokine respectively. β-actin was utilized for endogenous control of protein concentration in the samples and determined with the help of Human Actin Beta (ACTb) ELISA Kit (BMASSAY) based on the manufacturer’s recommendations. Furthermore, 100 μl of serum (ρprotein = 0.5 mg/ml) was added to pits coated with antibodies specific for the analyzed proteins and then incubated (1.5 hours, 37°C). Following incubation contents were removed and the pits were rinsed three times in 10 mM of phosphate buffered saline and re-incubated (1 hour, 37°C) with 100 μl of biotinylated antibodies specific for the analyzed proteins. Then, the content was removed and the pits were rinsed three times in 10 mM of phosphate buffered saline and incubated (30 minutes, 37°C) with 100 μl of ABC working solution. The content was again removed and the pits were rinsed five times in 10 mM of phosphate buffered saline and incubated (10 minutes, 37°C) with 90 μl of TMB substrate. After adding 100 μl of TMB Stop Solution, the absorbance of the samples was measured using Multiskan Ascent Microplate Photometer (Thermo Labsystems) at λ = 450 nm. In order to determine protein concentration, analytical curves for the proteins were created.
4
Quantifying Total Plasma Protein
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To assess Total protein concentration in blood plasma we used Micro BCATM Protein Assay Kit (ThermoSCIENTIFIC) based on the manufacturer’s recommendations. 150 µl of the reaction mixture was added to pits containing 150 µl of serum, diluted 10 times in 10 mM of phosphate buffered saline, pH 7.4, and incubated for2 hours, at 37°C. In order to measure protein concentration, an analytical curve for serum albumin was determined. Both the examined samples and the reference samples were positioned parallel in three repetitions. Eventually, Sample absorbance was measured using Multiskan Ascent Microplate Photometer (Thermo Labsystems) at λ = 570 nm and total protein concentration was calculated from the standard curve equation.
5
Serum Albumin and Total Protein Analysis
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An analytical curve for serum albumin was determined and the total protein concentration in the serum samples of the patients was measured. The analyzed samples and reference samples were carried out parallelly in three repetitions. Multiskan Ascent Microplate Photometer (Thermo Labsystems, Philadelphia, PA, USA) was used to quantify sample absorbance at λ = 562 nm; additionally, the standard curve equation made it possible to calculate total protein concentration.
6
Fucoidan Impacts Cell Viability and Death
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Cell viability was determined by a CCK-8 assay. In brief, Hca-F cells (1.0 × 105 cells/well, in 100 µL medium) seeded in 96-well microtiter plates were exposed to fucoidan at given concentrations for 6–48 h and treated simultaneously with CoCl2 in final volumes of 200 µL. Ten microliters of CCK-8 were added to each well and incubated for 30 min, and the absorbance at 490 nm was measured using a Multiskan Ascent microplate photometer (Thermo Fisher Scientific, Carlsbad, CA, USA). Cell viability was expressed as the percentage of the absorbance of treated cells relative to the absorbance of control cells. Similarly, cell death was identified by trypan blue staining for 24 h. The numbers of total cells and dead cells were counted. The cell death rate (D%) was calculated using the following equation: D% = (Cdeath cell/Ctotal cell) × 100%.
7
Evaluating 99mTc-RYM1 Binding in Lung Tissue
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Ex vivo binding of 99mTc-RYM1 to biological tissue was evaluated in lung tissue homogenate of CC10-IL-13Tg mice (n = 2) showing elevated basal MMP activity20 (link). Lung tissue (247 ± 85 µg, in duplicates was incubated with 3 nM of 99mTc-RYM1 for 60 min at 37 °C with or without the preincubation of 20 µM of RYM. After tracer incubation, tissue homogenate was spin down and washed 3 times with PBS, before resuspension in protein lysis buffer, gamma-counting (Wizard2, PerkinElmer) and protein concentration was measured (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific and Multiskan Ascent Microplate Photometer, Thermo Labsystems) in order to determine the amount of tissue used. All experimental protocols were approved by Yale University and VA Connecticut Institutional Animal Care and Use Committees.
8
Quantification of KIBRA and β-Actin Proteins
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The concentration of proteins KIBRA in the serum of the patients was determined using Human Kibra Elisa Kit (antibodies-online.com antibodies-online GmbH, Aachen, Germany) according to the protocols provided by the manufacturer. β-actin was used for endogenous control of protein concentration in the samples and determined with the help of Human Actin Beta (ACTb) ELISA Kit (BMASSAY) based on the manufacturer’s recommendations. 100 μl of serum (ρprotein=0.5 mg/ml) was added to pits coated with antibodies specific for the analyzed proteins and incubated (1.5 hours, 37°C). The content was removed and the pits were rinsed 3 times in 10 mM of phosphate buffered saline and incubated (1 hour, 37°C) with 100 μl of biotinylated antibodies specific for the analyzed proteins. Then, the content was removed and the pits were rinsed 3 times in 10 mM of phosphate buffered saline and incubated (30 minutes, 37°C) with 100 μl of ABC Working Solution. The content was removed and the pits were rinsed 5 times in 10 mM of phosphate buffered saline and incubated (10 minutes, 37°C) with 90 μl of TMB substrate. After adding 100 μl of TMB Stop Solution, the absorbance of the samples was measured using Multiskan Ascent Microplate Photometer (Thermo Labsystems) at λ=450 nm. In order to determine protein concentration, analytical curves for the analyzed proteins were made.
9
Serum Protein Quantification Protocol
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150 μl of the reaction mixture was added to pits containing 150 μl of serum, diluted 10 times in 10 mM of phosphate buffered saline, pH 7.4, and incubated (2 hours, 37°C). In order to specify protein concentration, an analytical curve for serum albumin was determined. Both the examined samples and the reference samples were made parallel in 3 repetitions. Sample absorbance was measured using Multiskan Ascent Microplate Photometer (Thermo Labsystems) at λ=562 nm and total protein concentration was calculated from the standard curve equation.
10
Quantifying Immune Biomarkers in Patients
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Serum concentration of IL-17, IL-21, IL-23, IL-35, and Foxp3 proteins was measured according to the manufacturers’ protocols with the use of Human IL-17 Quantikine ELISA Kit, Human IL-23 Quantikine ELISA Kit (R&D Systems, Inc. Minneapolis, MN, USA), Human IL21 ELISA Kit (CLIA) from LifeSpan BioSciences (Biocompare South San Francisco, CA, USA), Human IL-35 ELISA Kit and Human FOXP3 ELISA Kit (MyBioSource, Inc., San Diego, CA, USA). An endogenous control of protein concentration in the samples collected from the patients was performed with β-actin. Human Actin Beta (ACTb) ELISA Kit (BMASSAY) was used in this case in line with the recommendations specified by manufacturer. Multiskan Ascent Microplate Photometer (Thermo Labsystems) allowed the absorbance of the samples to be measured and analyzed at λ = 450 nm. Analytical curves for the proteins were prepared with the aim of determining protein concentration.
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