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Chemiluminescence assay

Manufactured by GE Healthcare
Sourced in United Kingdom

Chemiluminescence assay is a type of analytical technique used to measure the presence and quantity of specific molecules or analytes in a sample. It relies on the principle of chemiluminescence, where a chemical reaction generates light that can be detected and quantified. This method is commonly used in various fields, including clinical diagnostics, environmental analysis, and research applications.

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4 protocols using chemiluminescence assay

1

Western Blot Analysis of Liver Proteins

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Liver tissues were washed with ice-cold PBS and the protein extracts were prepared using ice-cold cell lyses buffer supplemented with protease inhibitor cocktail (IBI SCIENTIFIC, Peosta, USA). Protein concentrations were measured using Bradford assay (Bio-Rad, CA, USA) according to the manufacturer's protocol. Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels and transferred to nitrocellulose membrane. The membrane was blocked with 5% skimmed milk in TBS-T (10 mM Tris-HCl, 150 mM NaCl, 0.25% Tween 20, pH 7.5) at room temperature for 2 h followed by incubation with 2 μg/mL of primary antibody for GPx (sc-22145), CAT (sc-34285), GST (ab19256), PON-1 (sc-59646), PON-3 (sc-21156), PPAR-δ (ab8937), ABCA-1 (sc-58219), MCP-1 (sc-28879), and GAPDH (sc-32757) diluted in TBS and 5% skimmed milk overnight at 4°C. After washing with TBS-T buffer, the membrane was incubated with 1 μg/mL of horseradish peroxidase (HRP) labeled secondary antibody diluted in TBS-T buffer for 2 h at room temperature, followed by three washes with TBS-T buffer. The detection was performed using chemiluminescence assay (Amersham, GE Healthcare, Life Sciences, UK). Membranes were exposed to X-ray film to observe the bands (Kodak, Rochester, NY). Protein bands in treated and untreated (control) groups were quantified using the Kodak Scientific ID software.
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2

Protein Expression Analysis by Western Blot

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Cells were lysed in protein lysis buffer (Beyotime). The whole cell lysis products were analyzed with SDS-PAGE and then transferred to PVDF membrane (Millipore). The following primary antibodies were used: anti-KLF4 polyclonal antibody (ab106629, Abcam), anti-DMP1 polyclonal antibody (ab103203, Abcam), anti-DSP polyclonal antibody (NBP191612, NOVUS), and anti-β-ACTIN monoclonal antibody (660091Ig, Proteintech). After incubation with the corresponding antibodies, the membrane was washed 3 times for 5 min each with TBST. We used ImageJ software for further densitometric analysis. Bound primary antibodies were detected by incubating for 1 h with horseradish peroxidase conjugated goat anti-mouse or anti-rabbit IgG (Biofly, China) for analysis. The membrane was washed and developed by a chemiluminescence assay (GE Healthcare).
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3

Whole Cell Lysate Preparation for p73 Analysis

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To prepare whole cell lysates, cells were collected by centrifugation and washed once with PBS and then lysed in EBC lysis buffer (Cold Spring Harb Protoc; 2006; doi:10.1101/pdb.rec8856) [50 mM Tris (pH 8.0), 120 mM NaCl, 0.5% Nonidet P-40] supplemented with aprotinin (11.5 μg/ml), leupeptin (11.5 μg/ml) phenylmethylsulfonyl fluoride (50 μg/ml), NaF (100 mM) and Na ortovanadate (0.2 mM). Protein concentration was determined by BCA following the manufacturer's instructions (G Biosciences). Proteins (25 μg) were resolved in SDS/PAGE and transferred to PVDF filters. Blots were incubated with a mouse antibody against p73 (ER-15) (Thermo-Pierce) and then incubated with an anti-mouse antibody conjugated with horseradish peroxidase (Santa Cruz). Bound antibody was detected by a chemiluminescence assay (GE Healthcare).
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4

Western Blot Analysis of Osteogenesis Regulators

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Cells were lysed in NP40 Lysis Buffer with protease inhibitor cocktail (MCE). The cell lysis were analyzed with electrophoresis and then transferred to PVDF membrane (Millipore). The filter was blocked for 1-2 hours by 5% nonfat milk in TBST (Tris buffered saline containing Tween 20) and then incubated with the primary antibodies at 4 °C overnight. The primary antibodies used were: anti-IPO7 monoclonal antibody (sc365231, Santa Cruz), anti-RUNX2 polyclonal antibody (YT5356, Immunoway), anti-KLF4 polyclonal antibody (4038, Cell Signaling technology), anti-Phospho-SMAD1/5 monoclonal antibody (9516, Cell Signaling technology), anti-OSX monoclonal antibody (sc393325, Santa Cruz), anti-DLX3 polyclonal antibody (132613AP, Proteintech), anti-DMP1 polyclonal antibody (ab103203, Abcam), anti-DSP polyclonal antibody (NBP191612, NOVUS), anti-β-ACTIN monoclonal antibody (660091Ig, Proteintech) and anti-PCNA polyclonal antibody (102052AP, Proteintech). After incubation with the corresponding antibodies, the filter was washed 3 times for 5 minutes each with TBST. The filter was then incubated with HRP (horseradish peroxidase) conjugated goat anti-Mouse or anti-Rabbit IgG (Biofly, China) for 1 hour. The filter was washed and developed by a chemiluminescence assay (GE Healthcare). We used ImageJ software for further analysis.
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