Cell lysates and Western blots were prepared as previously described [20 (link)]. Following antibodies and concentrations/dilutions were used: rabbit anti-PUMA (1 μg/ml)/1:1000; rabbit anti-cleaved CASP-9 (1 μg/ml)/1:1000; rabbit anti Cleaved poly-ADP-ribose polymerase-1 (PARP-1)/1∶1000 (Cell Signaling; Frankfurt, Germany); purified mouse anti-human p53 (0.5 mg/ml)/1:100 (clone DO-7; BD Pharmingen; Schwechat, Austria); mouse anti-p21WAF1 (0.5 μg/ml)/1:2000 (Zymed; San Francisco, CA, USA); polyclonal rabbit anti-human phospho-mTOR (Ser2448) and mTOR Antibody (#2972) Antibody (Cell signaling; Frankfurt, Germany). As secondary antibodies, we used rabbit anti-mouse and swine anti-rabbit HRP-conjugated antibodies at a final concentration of 1 μg/ml/1:1000 (DAKO; Copenhagen, Denmark). Specific protein bands were visualized by enhanced chemiluminescence assay (Amersham Biosciences; Buckinghamshire, England). All Western blots were probed with a polyclonal rabbit anti β-tubulin antibody (Anti-TubA8/1:1000) (Sigma-Aldrich, Vienna, Austria) to demonstrate equal protein loading. Protein expression was quantified using Image J software.
Enhanced chemiluminescence assay
Manufactured by Cytiva
Sourced in United Kingdom
Enhanced chemiluminescence assay is a laboratory technique used to detect and quantify specific proteins or molecules in a sample. It is a sensitive method that utilizes the emission of light during a chemical reaction to generate a signal that can be measured and analyzed.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Las 4000 image analyzer, by Fujifilm (1 mentions)
Non fat milk, by Nestlé (1 mentions)
Mmp 12 h 52, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Blotting grade non fat dry milk, by Bio-Rad (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Anti caspase 12, by Abcam (1 mentions)
Mouse antihuman bcl xl, by Santa Cruz Biotechnology (1 mentions)
Mouse anti human bcl 2, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Bio-Rad (1 mentions)
Las 3000, by Fujifilm (1 mentions)
5 protocols using enhanced chemiluminescence assay
1
Western Blot Analysis of Apoptosis Markers
2
Western Blot Analysis of Phosphorylated IRE1
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Cells were lysed with lysis buffer containing 50 mM Tris pH 7.2, 150 mM NaCl, 1 mM EDTA, and 1% triton X-100 containing protease inhibitors (1 mM PMSF, 10 μg/mL aprotinin, and 1 mM Na3VO4). For detection p-IRE1, SDS was added to lysis buffer to a final concentration of 0.5%. The protein concentration was assayed by the BCA protein assay kit (Pierce, Rockford, IL). Equal amount of protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. Membranes were immunoblotted with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Protein signals were visualized by an enhanced chemiluminescence assay (Amersham, Piscataway, NJ). The chemiluminescence photographs were taken with LAS-4000 Image Analyzer (Fujifilm, Japan) and processed by Adobe Photoshop 7.0 (Adobe System Inc., San Jose, CA).
3
Quantifying MMP-12 in Lung Homogenate
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Total protein (30 µg/lane) from lung homogenate was subjected to 8 or 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were then washed once with Tris/HCl, pH 7.4, containing 159 mM NaCl and 1% Tween 20, Tris buffered saline (TBS), and then blocked with 5% non-fat milk (Nestlé, São Paulo, Brazil). After, the membranes were washed with TBS and probed with MMP-12 (H-52; Santa Cruz Biotechnologies, Santa Cruz, CA, USA). β-actin (#5441; Sigma-Aldrich, St. Louis, MO, USA; 1:1000) was used as a loading control. After washing with TBS-T, following incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:2000), after washing with TBS, immunoreactive protein bands were revealed using an enhanced chemiluminescence assay (Amersham Pharmacia, Little Chalfont, UK). The results are expressed as arbitrary units.
4
Ozone Exposure Impacts on Hippocampal Protein Expression
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The hippocampus of each of the groups exposed to ozone were homogenized, and SDS-PAGE was performed according to the Laemmli method. The proteins were separated in a 10% polyacrylamide gel under denaturing conditions. After the run, the proteins were electrotransferred to a polyvinylidene difluoride membrane (Immobilon P; Millipore) in a semidry electroblotting system (Bio-Rad) at 25 V for 50 min. Membranes were blocked by incubation in a buffer containing 20 mM Tris base, pH 7.5, 500 mM NaCl, 0.05% Tween-20 (TBS-T buffer) and 5% blotting grade nonfat-dry milk (BioRad). Then, they were tested with ATF6 (1:500, ABCAM ab11909), GRP/78 (1:900, ABCAM ab21685), anti-caspase 12 (1:500, ABCAM ab62484) or GAPDH (1:10,000, Cell Signaling #2118). The membranes were incubated for 2 h with the secondary antibody at an adequate dilution in the TBS-T buffer. After three washes with TBS-T buffer, bands were visualized using horseradish peroxidase-conjugated goat anti-rabbit IgG (Pierce) at a dilution of 1:10,000 and using the Enhanced ChemiLuminescence assay (Amersham Life Science) according to the manufacturer’s instructions. ATF6, GRP/78 and caspase 12 were normalized using anti-GAPDH as loading control to confirm equal amounts of protein.
5
Evaluating BCL-XL and BCL-2 Expression in Transduced Cells
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Transduced cells, including UT-7/Epo cells, were collected at 24, 48, and 72 hours. Cells were lysed with lysis buffer containing 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, and 1.0% NP-40. The protein concentration was determined using the Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL). Whole-cell extracts (5 mg/lane) were subjected to 12.5% sodium dodecyl sulphate (SDS) -polyacrylamide gels, and protein was transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA). The immunoreaction was performed by incubating the membrane for 1 hour at room temperature (RT) with primary antibodies as follows: mouse antihuman BCL-XL (Santa Cruz Biotechnology, Santa Cruz, CA; dilution, 1:200), mouse antihuman BCL-2 (Santa Cruz Biotechnology; dilution, 1:200), or mouse anti-b-actin (C4, sc-47778, Santa Cruz Biotechnology; dilution, 1:1,000). Membranes were incubated at RT for 1 hour with horseradish peroxidase (HRP) -conjugated secondary antibody: antimouse immunoglobulin G antibody (IgG Ab) (sc-2005, Santa Cruz Biotechnology; dilution of 1:10,000). Antigen-antibody reactions were detected using the enhanced chemiluminescence assay (Amersham Biosciences, Piscataway, NJ). Western blots were analyzed on an LAS3000 (Fuji Film Co., Tokyo, Japan).
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