Enrichment of low-abundance plasma proteins using the CPLL column (ProteoMiner; #163-3006, Bio-Rad Laboratories, Inc., CA, USA) was performed according to the manufacturer's instructions. Briefly, the CPLL column was prepared by adding 200 μl wash buffer (BioRad) and rotating the column several times over a 5 min period. The wash buffer was removed by centrifugation at 1,000 × g for 1 min. This step was repeated once. Thereafter, 200 μl of plasma was added to the column followed by incubation for 2 h at room temperature with gentle mixing. The unbound proteins were then removed by 1000 x g centrifugation for 1 min, and the column was washed twice using 200 μL wash buffer (BioRad) and additionally washed by 200 μL deionized water to remove unbound proteins and salt contamination. The bound proteins were eluted by adding 20 μl of elution reagent (BioRad) and then incubation for 15 min with intermittent gentle mixing. The eluted proteins were collected by centrifugation at 1,000 × g for 30-60 sec. This elution step was repeated twice. The eluate was kept at -80°C until further analysis.
Wash buffer
Manufactured by Bio-Rad
Sourced in United States
Wash buffer is a solution used in various laboratory techniques to remove unwanted materials or impurities from samples or surfaces. It serves the core function of facilitating the cleaning and preparation of samples for further analysis or experiments.
Automatically generated - may contain errors
4 protocols using wash buffer
1
Plasma Proteome Enrichment Using CPLL
2
Multiplex Cytokine Profiling of Plasma
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The plasma samples obtained from the 24-hour post-injury groups in Cohort II (n = 8 per group) were used for a multiplex assay to compare cytokines, chemokines and growth factors, as per manufacturer protocol (Bio-Rad, Bio-Plex Pro™ Rat Cytokine 23-Plex Assay #L8001V11S5). Briefly, plasma samples were diluted 1:4 and analysed following the manufacturer's instructions. The plates were pre-wetted with wash buffer (Bio-Rad) and loaded with 1 × beads (Bio-Rad). The wells were rinsed by wash buffer (Bio-Rad) and then loaded with blank, standards, or samples for one-hour incubation. Following washing thre times, the detection antibodies were added for 30-minute incubation. The plates were washed and incubated in streptavidin-phycoerythrin for 10 minutes. After washing, the analytes were resuspended with assay buffer (Bio-Rad) for 30 seconds. The plates were read using the Bio-Plex reader (Bio-Rad), and results were converted to a pg/ml concentration for all of the cytokines tested.
3
Multiplexed Immunoassay Protocol
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About 50 μl 1x beads were added to a 96 well micro titer plate and washed 2x with the Bio-Rad wash buffer, (Cat #, 10,014,939). The standards, samples and controls were diluted (Bio-Rad Antibody diluent, (Cat #, 35,002,989) and 50 μl added to the wells and incubated in the dark at room temperature with shaking at 850 rpm for 1 h. After incubating the beads, samples, standards, blank and controls; the plates were washed three times with 100 μl wash buffer. 1x detection antibodies were added to the assay plate and incubated in the dark for 30 min with shaking at 850 rpm. The plate was washed 3x and Streptavidin – PE (Cat #, L9703897) was added. The assay plate was incubated for 10 min at room temperature with shaking at 850 rpm. The plate was washed 3x and the beads (Bio-Rad Cat #, 171–304,040) were re-suspended for plate reading. The Bio-plex manager software was used running the assay, data acquisition and analysis.
4
Cytokine and Chemokine Quantification in Rat Colon
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Rats were anesthetized with pentobarbital (50 mg/kg) and killed by decapitation. The 2 cm length colon was removed starting from anus, washed with wash buffer (Bio-Rad Laboratories, Inc., Hercules, California, USA), and homogenized in Bio-Plex cell lysis buffer (Bio-Rad Laboratories, Inc., Hercules, California, USA). After centrifugation, the supernatants were collected and stored at −80°C until further use. The supernatants were used to assay cytokines and chemokines using Bio-Plex Pro Rat Cytokine 24-Plex Assay kit (Bio-Rad Laboratories, Inc., Hercules, California, USA). The following cytokines and chemokines were quantified: interleukin- (IL-) 1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-17, IL-18, erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), growth-related oncogene/keratinocyte-derived chemokine (GRO/KC), interferon-γ (IFN-γ), macrophage colony-stimulating factor (M-CSF), monocyte inflammatory protein-1α (MIP-1α), MIP-3α, regulated on activation normal T cell expressed and secreted (RANTES), tumor necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein (MCP-1).
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