Cultured cells were serum starved for 48 h prior to stimulation. VSMCs were stimulated for 1 to 30 min with SAA and OxPAPC. The antagonist was pre-incubated for 30 minutes. For the quantification of phosphorylated proteins, cells were washed with ice-cold PBS and lyzed using a buffer included in the kit (Bio-Plex® Cell Lysis Kit). Total content of IkB-a and phosphorylated IkB-a (p-IkB-a) was measured with the Bio-Plex® Phosphoprotein Detection Kit (BioRad, Muenchen, Germany) according to manufacturer´s instruction using Luminex™ technique (BioRad, Muenchen, Germany). The phosphorylated protein was normalized to the total protein content of the lyzed cells.
Bio plex phosphoprotein detection kit
Manufactured by Bio-Rad
Sourced in Germany, United States
The Bio-Plex® Phosphoprotein Detection Kit is a tool designed to quantify the levels of phosphorylated proteins in biological samples. It utilizes a multiplex assay format to simultaneously measure multiple phosphoproteins in a single well. The kit is compatible with the Bio-Plex® suspension array system.
Automatically generated - may contain errors
Lab products found in correlation
Bio plex manager software, by Bio-Rad (2 mentions)
Cell lysis kit, by Bio-Rad (1 mentions)
Luminex technique, by Bio-Rad (1 mentions)
Bio plex pro 2 wash station, by Bio-Rad (1 mentions)
Phosphate buffered saline (pbs), by PAN Biotech (1 mentions)
Bio plex 200 system, by Bio-Rad (1 mentions)
Bio plex manager 6, by Bio-Rad (1 mentions)
Bio plex 200 array system, by Bio-Rad (1 mentions)
Bio plex pro assay kit, by Bio-Rad (1 mentions)
5 protocols using bio plex phosphoprotein detection kit
1
Quantifying Intracellular Signaling Dynamics
2
Multiplex Bead-Based Immunoassay for Phosphoproteins
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A commercially available multiplex bead-based immunoassay kit (Bio-Plex Phosphoprotein Detection Kit, Bio-Rad Laboratories) was used according to the manufacturer’s protocol to detect the phosphorylation of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) and C-Jun N-terminal kinase (JNK), protein kinase B (Akt), and nuclear factor kappa B (NF-kB) in lysates from purified T lymphocytes obtained from SLE patients treated as described above. Data were analyzed with Bio-Plex manager software, version 4.1.1 (Bio-Rad Laboratories) and reported as fluorescence intensity (FI). Values with a coefficient of variation >12 % were excluded.
3
Hepatocyte pAkt S473 phosphorylation assay
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Hepatocytes were seeded at confluence (2 × 106 cells/6-cm dish) in full medium as described above, and following cell adhesion were washed with PBS (PAN Biotech) and subsequently cultivated in serum-free cultivation medium (phenol red-free Williams E medium supplemented with 2 mM l -glutamine and 1% (v/v) penicillin/streptomycin 100×) for 6 h prior to 10 min stimulation with the indicated doses of rmHGF. To stop stimulation, the hepatocytes were treated as described above. Supernatants constituting total cellular lysates were incubated over night with beads coupled with pAkt S473 antibody (Bio-Rad) and assayed with the Bioplex phosphoprotein detection kit (Bio-Rad). Washing procedures were performed using the Bio-Plex Pro™ II Wash Station (Bio-Rad). The pAkt S473 fluorescence intensity of the analyzed samples was acquired using the Bio-Plex Pro™ II instrument (Bio-Rad).
4
Phosphoprotein Profiling in Neurons
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At specific time points, neurons were collected, and protein lysates were prepared, and content of phosphorylated proteins was detected using the respective Bio-Plex Phosphoprotein Detection kit (Bio-Rad, USA) according to the manufacturer's protocol. Briefly, 50 μl of cell lysate (adjusted to a concentration of 0.6–0.9 μg/ml) was plated in the 96-well filter plate coated with beads coupled to anti-phosphoprotein antibody. The plate was incubated overnight at room temperature on a platform shaker. After a series of washes to remove unbound proteins, biotinylated detection antibodies, each specific for a different epitope, were added to the reaction. This resulted in formation of antibody complexes assembled around the target proteins. Streptavidin-phycoerythrin was then added to bind to the biotinylated detection antibodies on the bead surface. Data were acquired with the Bio-Plex 200 system and analyzed with the Bio-Plex Manager software (Bio-Rad).
5
Cytokine and Phospho-Protein Profiling
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Cell-free culture supernatants of human monocytes (50 ml) were assayed for the presence of the indicated cytokines, chemokines, and growth factors using the Bio-Plex Pro assay kit (Bio-Rad). For phospho-IkBa protein, monocyte protein extracts were prepared and analyzed using the Bio-Plex phospho-protein detection kit (Bio-Rad) as per the manufacturer's instructions. All data were collected using the Bio-Plex 200 array system with Luminex xMap Technology and analyzed using Bio-Plex Manager 6.0 (Bio-Rad).
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