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Western light chemiluminescent detection system

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The Western-Light chemiluminescent detection system is a laboratory instrument designed to detect and analyze protein expression levels in Western blot experiments. It utilizes chemiluminescent technology to generate and capture light signals emitted during the detection process, allowing for sensitive and quantitative analysis of target proteins.

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Lab products found in correlation

Bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Pvdf microporous membrane, by Merck Group (2 mentions) Biorad transblot sd semi dry transfer cell, by Bio-Rad (2 mentions) Proprietary luminescent substrate, by Thermo Fisher Scientific (2 mentions) Nitrocellulose blotting membrane, by Merck Group (1 mentions) Cell lysis buffer, by Cell Signaling Technology (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) 0.2 μm nitrocellulose membrane, by GE Healthcare (1 mentions) 0.2 m nitrocellulose membrane, by GE Healthcare (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Protease inhibitor, by Keygen Biotech (1 mentions) Phosphatase inhibitor, by Keygen Biotech (1 mentions) Phenylmethylsulfonyl fluoride, by Keygen Biotech (1 mentions) Nitrocellulose paper, by Bio-Rad (1 mentions) Protease phosphatase inhibitor cocktail, by Roche (1 mentions)

15 protocols using western light chemiluminescent detection system

1

Western Blot Analysis of Cellular Proteins

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After collection, the cells were lysed in a cell lysis buffer (#9803, Cell Signaling Technology, Beverly, MA, USA) mixed with protease inhibitor cocktail (Roche, Indianapolis, IN, USA). The cell lysates were quantified and the equal concentration of proteins were loaded and analyzed in 10% SDS-PAGE. The separated proteins were transferred onto a nitrocellulose blotting membrane (Millipore, Billerica, MA, USA). Then, the membrane was blocked with non-fat milk and probed with the indicated primary antibodies and secondary antibodies. The blotting bands were detected using the Western-Light chemiluminescent detection system (Applied Biosystems, Foster, CA, USA).
Wang T.C., Luo S.J, & Chang S.F. (2021). Bone Morphogenetic Protein 7 Effect on Human Glioblastoma Cell Transmigration and Migration. Life, 11(7), 708.
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2

Evaluating NF-κΒ Phosphorylation and Expression

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To assess phosphorylation of the p65 subunit of NF-κΒ and expression levels of ΝF-κΒ, MDCK cells were seeded at a concentration of 2 × 105 cells/well (24-well plates) for 24 h and then infected or not for 24 h with intracellular L.m. at a MOI~200 bacteria/cell. Cells in infected wells were treated with vehicle control or the NEMO binding peptide (50 μM) starting 4 h p.i.. Cells were then lysed with a buffer containing 0.5M Tris-HCl, pH 7.4, 1.5M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10mM EDTA and a protease inhibitor mixture (phenylmethylsulfonyl fluoride [PMSF], leupeptin, aprotinin, and sodium orthovanadate). The total cell lysate was separated by SDS–PAGE (10% running, 4% stacking) and transferred onto a nitrocellulose membrane (Immobilon P, 0.45-μm pore size). The membrane was then incubated with the designated antibodies. Immunodetection was performed using the Western-Light chemiluminescent detection system (Applied Biosystems). Membrane blots from 76 kDa to 52 kDa were imaged for phospho-p65 and p65 (65 kDa) and 52 kDa to 33 kDa were imaged for the corresponding β-actin (42 kDa) (Figure S6F).
Bastounis E.E., Serrano-Alcalde F., Radhakrishnan P., Engström P., Gómez-Benito M.J., Oswald M.S., Yeh Y.T., Smith J.G., Welch M.D., García-Aznar J.M, & Theriot J.A. (2021). Mechanical competition triggered by innate immune signaling drives the collective extrusion of bacterially-infected epithelial cells. Developmental cell, 56(4), 443-460.e11.
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3

Mechanosensitive Protein Phosphorylation

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To assess phosphorylation and expression levels of different mechanosensitive proteins28 (link), cells were seeded at a concentration of 2 × 105 cells/well (24-well plates) on soft 3 kPa or stiff 70 kPa hydrogels for 24 h, and then lysed with a buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, and a protease inhibitor mixture (phenylmethylsulfonyl fluoride [PMSF], leupeptin, aprotinin, and sodium orthovanadate). The total cell lysate was separated by SDS–PAGE (10% running, 4% stacking) and transferred onto a nitrocellulose membrane (Immobilon P, 0.45-μm pore size). The membrane was then incubated with the designated antibodies. Immunodetection was performed using the Western-Light chemiluminescent detection system (Applied Biosystems).
Bastounis E.E., Yeh Y.T, & Theriot J.A. (2019). Subendothelial stiffness alters endothelial cell traction force generation while exerting a minimal effect on the transcriptome. Scientific Reports, 9, 18209.
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4

Western Blot Analysis of COL1 and ALP

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Cultured/treated cells were lysed with the lysis buffer containing protease and phosphatase inhibitors (phenylmethylsulfonylfluoride, aprotinin, and sodium orthovanadate). Total protein from the cell lysate (100 µg of protein) was separated using SDS-PAGE (using a 10% running, and 4% stacking polyacrylamide gel) and separated proteins were transferred onto a nitrocellulose membrane (Immobilon P; 0.45-µm pore size). The blot was then treated with the indicated antibodies. Chemiluminescent bands were detected by using the Western-Light chemiluminescent detection system (Applied Biosystems). COL1 and ALP expression levels were normalized with those of ß-actin internal control from the same sample.
Huang K.C., Chuang P.Y., Yang T.Y., Huang T.W, & Chang S.F. (2019). Hyperglycemia inhibits osteoblastogenesis of rat bone marrow stromal cells via activation of the Notch2 signaling pathway. International Journal of Medical Sciences, 16(5), 696-703.
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5

Western Blot Protein Detection

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Cells were collected and lysed with a RIPA buffer containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a protease inhibitor mixture (PMSF, aprotinin, and sodium orthovanadate). The total cell lysate was separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was then incubated with the designated antibodies. Immunodetection was performed by using the Western-Light chemiluminescent detection system (Applied Biosystems, Foster City, CA).
Chang S.F., Hsieh R.Z., Huang K.C., Chang C.A., Chiu F.Y., Kuo H.C., Chen C.N, & Su Y.P. (2015). Upregulation of Bone Morphogenetic Protein-2 Synthesis and Consequent Collagen II Expression in Leptin-stimulated Human Chondrocytes. PLoS ONE, 10(12), e0144252.
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6

FAK and Met Phosphorylation Assessment

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To assess FAK and Met phosphorylation and expression levels, cells were seeded on different substrates for 24 h, and then lysed with a buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, and a protease inhibitor mixture (phenylmethylsulfonyl fluoride [PMSF], leupeptin, aprotinin, and sodium orthovanadate). The total cell lysate was separated by SDS–PAGE (10% running, 4% stacking) and transferred onto a nitrocellulose membrane (Immobilon P, 0.45-µm pore size). The membrane was then incubated with the designated antibodies. Immunodetection was performed using the Western-Light chemiluminescent detection system (Applied Biosystems).
Bastounis E.E., Yeh Y.T, & Theriot J.A. (2018). Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin. Molecular Biology of the Cell, 29(13), 1571-1589.
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7

Protein Isolation and Western Blot Analysis

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Cells were lysed in 1% OG buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% OG, 1 mM EDTA, 10 μg/ml leupeptin, 2 μg/ml aprotinin and 1 mM PMSF). A BCA Protein Assay Kit (Pierce Biotechnology, Rockford, Illinois) was then used to determine the total protein concentration, and equal amounts of protein were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Billerica, MA). The membrane was blocked with 5% non-fat milk and then incubated for 2 h at room temperature with the designated antibody. The Western-Light chemiluminescent detection system (Applied Biosystems, Foster, CA) was used for immunodetection.
Tang J., Guo Y.S., Yu X.L., Huang W., Zheng M., Zhou Y.H., Nan G., Wang J.C., Yang H.J., Yu J.M., Jiang J.L, & Chen Z.N. (2015). CD147 reinforces [Ca2+]i oscillations and promotes oncogenic progression in hepatocellular carcinoma. Oncotarget, 6(33), 34831-34845.
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8

Investigating C70PLY4 Regulation of ERK1/2 and NFκB

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Cells were kept untreated as the control, treated with LPS (1 μg/mL), or co-treated with LPS (1 μg/mL) and C70PLY4 (100, 300, and 500 nM) for 24 h, and the phosphorylation of the ERK1/2 and NFκB-p65 subunits was determined by Western blot. Cells were collected by scraping and lysed with RIPA buffer containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a protease inhibitor cocktail. The total cell lysate (50 µg of protein) was separated by SDS-polyacrylamide gel electrophoresis (PAGE) (10% running, 4% stacking) and transferred onto a polyvinylidene fluoride membrane (Immobilon P, 0.45 µm pore size). The membrane was then incubated with the designated antibodies. Immunodetection was performed using the Western-Light chemiluminescent detection system (Applied Biosystems, Foster City, CA, USA).
Chang S.F., Chen C.N., Lin J.C., Wang H.E., Mori S., Li J.J., Yen C.K., Hsu C.Y., Fung C.P., Chong P.C., Leng C.H., Ding Y.J., Chang F.Y, & Siu L.K. (2020). Truncated Pneumolysin from Streptococcus pneumoniae as a TLR4-Antagonizing New Drug for Chronic Inflammatory Conditions. Cells, 9(5), 1183.
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9

Western Blot Analysis of Proteoglycans

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For Western immunoblotting, proteoglycans isolated by ion-exchange chromatography were digested with 0.05 units chondroitin ABC lyase (North Star BioProducts, East Falmouth, MA) in 0.3 M Tris-HCl, pH 8.0, 0.6 mg/ml bovine serum albumin and 18 mM sodium acetate with protease inhibitors for 3 h at 37 °C) and run on SDS-PAGE (4–12% with 3.5% stacking gel) under reducing conditions (Laemmli 1970 (link)). Separated proteins were electrophoretically transferred to 0.2 μm nitrocellulose membranes (GE Healthcare, Piscataway, NJ) using a BioRad Transblot SD Semi-Dry Transfer Cell (BioRad, Hercules, CA) (Olin et al. 1999 (link)). The transferred proteins were then detected with the primary antibody to versican (Millipore), and enhanced chemiluminescence (Western-Light Chemiluminescent Detection System) with proprietary luminescent substrate (Applied Biosystems, Foster City, CA) (Lemire et al. 2007 (link)).
Chang M.Y., Tanino Y., Vidova V., Kinsella M.G., Chan C.K., Johnson P.Y., Wight T.N, & Frevert C.W. (2014). A Rapid Increase in Macrophage-Derived Versican and Hyaluronan in Infectious Lung Disease. Matrix biology : journal of the International Society for Matrix Biology, 34, 1-12.
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10

Immunoprecipitation and Western Blotting

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Cells were washed and resuspended in NPBS and then lysed in 1% OG buffer. A BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) was then used to determine the total protein density. Lysates (1 mL) were immunoprecipitated by incubation with 2 μg of the designated antibody and 25 μL of protein A-agarose overnight at 4°C, followed by Western blotting with the designated antibody for 2 h at room temperature. The Western-Light chemiluminescent detection system (Applied Biosystems, Foster City, CA, USA) was used for immunodetection.
Tang J., Guo Y.S., Yu X.L., Huang W., Zheng M., Zhou Y.H., Nan G., Wang J.C., Yang H.J., Yu J.M., Jiang J.L, & Chen Z.N. (2015). CD147 reinforces [Ca2+]i oscillations and promotes oncogenic progression in hepatocellular carcinoma. Oncotarget, 6(33), 34831-34845.
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