The In-Cell Western assay is a proven method for the rapid quantification of proteins in cells [28 (link), 29 (link)]. BV-2 microglia were seeded into a 96-well plate at 5 × 104 cells/ml, and cells treated at 70% confluence. At the end of each experiment, cells were fixed with 8% formaldehyde solution (100 µL) for 15 min, followed by washing with PBS. The cells were then incubated with primary antibodies overnight at 4 °C. The following antibodies were used: rabbit anti-iNOS (Abcam), rabbit anti-phospho-p65 (Cell Signalling Technology), rabbit anti-phospho-IκBα (Santa Cruz Biotechnology), rabbit anti-NLRP3 (Abcam) and rabbit anti-phospho-p38 (Cell Signalling Technology) antibodies. Thereafter, cells were washed with PBS and incubated with anti-rabbit HRP secondary antibody for 2 h at room temperature. Then, 100 µl of HRP substrate was added to each well and absorbance measured at 450 nm with a Tecan Infinite M microplate reader. Readings were normalised with Janus Green normalisation stain (Abcam).
Janus green normalisation stain
Manufactured by Abcam
Janus Green normalisation stain is a laboratory reagent used to stain cellular structures and organelles for microscopic analysis. It provides a consistent and uniform staining pattern, allowing for the normalization of samples during image analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti phospho p65, by Cell Signaling Technology (3 mentions)
Rabbit anti phospho p38, by Cell Signaling Technology (2 mentions)
Rabbit anti phospho iκbα, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti nlrp3, by Abcam (2 mentions)
Infinite m microplate reader, by Tecan (2 mentions)
Rabbit anti inos, by Abcam (1 mentions)
Rabbit anti total iκbα, by Santa Cruz Biotechnology (1 mentions)
Infinite m nano microplate reader, by Tecan (1 mentions)
3 protocols using janus green normalisation stain
1
Quantification of Cellular Proteins via In-Cell Western
2
Quantifying Intracellular Protein Signaling
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The in-cell western (cytoblot) analysis is a proven method for the rapid quantification of proteins in cells [18 (link), 19 (link)]. PBMCs were seeded into a black 96-well plate at 5 × 104 cells/mL. At 70% confluence, cells were stimulated with spike glycoprotein S1 (100 ng/ml) for different periods. At the end of each experiment, cells were fixed with 8% paraformaldehyde solution (100 μL) for 15 min, followed by washing with PBS. The cells were then incubated with primary antibodies overnight at 4 °C. The following antibodies were used: rabbit anti-phospho-p65 (Cell Signalling Technology), rabbit anti-phospho-IκBα (Santa Cruz Biotechnology), rabbit anti-total IκBα (Santa Cruz Biotechnology), rabbit anti-phospho-p38 (Cell Signalling Technology) and rabbit anti-NLRP3 (Abcam) antibodies. Thereafter, cells were washed with PBS and incubated with anti-rabbit HRP secondary antibody for 2 h at room temperature. Then, 100 μL HRP substrate was added to each well and absorbance measured at 450 nm with a Tecan Infinite M microplate reader. Readings were normalised with Janus Green normalisation stain (Abcam).
3
Evaluation of G. kola Extract and Garcinoic Acid on NF-κB Signaling
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PBMCs were seeded into a 96-well plate at 5 × 10 4 cells/mL and allowed to settle overnight. Cells were then treated with either G. kola extract (6.25, 12.5 and 25 µg/mL) or garcinoic acid (1.25, 2.5 and 5 µM) and incubated for 1 h prior to stimulation with spike protein S1 (100 ng/mL) for a further 15 min. At the end of each experiment, cells were fixed with 8% paraformaldehyde solution (100 L) for 15 min., followed by washing with PBS. The cells were then incubated with primary antibodies overnight at 4°C. The following antibodies were used: rabbit antiphospho-p65 (Cell Signalling Technology), rabbit anti-IB (Cell Signalling Technology), and rabbit anti-phospho-IB (Santa Cruz Biotechnology) antibodies.
Thereafter, cells were washed with PBS and incubated with anti-rabbit HRP secondary antibody for 2 h at room temperature. Then, 100 µL HRP substrate was added to each well and absorbance measured at 450nm with a Tecan Infinite M Nano microplate reader. Readings were normalised with Janus Green normalisation stain (Abcam).
Thereafter, cells were washed with PBS and incubated with anti-rabbit HRP secondary antibody for 2 h at room temperature. Then, 100 µL HRP substrate was added to each well and absorbance measured at 450nm with a Tecan Infinite M Nano microplate reader. Readings were normalised with Janus Green normalisation stain (Abcam).
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