For Western immunoblotting, cultured SCs were homogenized in the modified RIPA buffer. Lysates were fractionated on a SDS-PAGE gel and transferred to a nitrocellulose membrane (Millipore). The blotted membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) at room temperature for 1 hr, and the membranes were incubated overnight at 4 °C with primary antibodies diluted in TBST containing 3% nonfat milk. After 3 washes in TBST, the blots were reacted with HRP-conjugated secondary antibodies for 1 hr at room temperature and then washed again with TBST. For detection, an enhanced chemiluminescence-Western blot system (Amersham, Piscataway, USA) was employed and images were analyzed with LuminoGraph III (ATTO, Tokyo, Japan). The relative gray value of each target protein (gray value of a target band/gray value of β-actin band) was calculated with software CS analyzer (ATTO, Tokyo, Japan). For the quantitative analysis, at least three independent experiments were performed.
Enhanced chemiluminescence western blot system
Manufactured by Cytiva
Sourced in United States
The Enhanced chemiluminescence-Western blot system is a laboratory equipment designed for the detection and quantification of proteins using Western blot analysis. It provides a sensitive and reliable method for visualizing and analyzing target proteins in complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (2 mentions)
Las image analysis system, by Fujifilm (2 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Horseradish peroxidase conjugated secondary antibodies, by Abmart (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Anti his tag antibody, by Abcam (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Il 1β, by Abcam (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Il 1α, by Santa Cruz Biotechnology (1 mentions)
6 protocols using enhanced chemiluminescence western blot system
1
Western Blot Analysis of Cultured SCs
2
Quantitative Analysis of GPRC5B Expression
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For Western blot analyses, cultured sciatic explants were prepared and thirty-five micrograms of total protein was separated and transferred to a nitrocellulose membrane (Amersham Bioscience, Piscataway, NJ, USA). The blots were reacted with the proper primary antibody (against GPRC5B or β-actin as control) and with horseradish peroxidase-conjugated secondary antibody. Detection was performed utilizing an enhanced chemiluminescence-Western blot system (Amersham Bioscience). LAS image-analysis system (Fujifilm, Tokyo, Japan) was used to quantification. All experiments were repeated a minimum of three times.
3
Western Blot Analysis of Protein Expression
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For Western immunoblotting, cultured cells were homogenized in lysis buffer (0.25 M Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 10% mercaptoethanol). The protein lysates were fractionated on a SDS-PAGE gel and transferred to nitrocellulose membrane (Millipore, USA). The blotted membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 1 hour, and the membranes were incubated overnight at 4°C with primary antibodies diluted in prepared TBST containing 3% nonfat milk. After 3 washes in TBST, the blots were reacted with horseradish peroxidase-conjugated secondary antibodies (Abmart, China) for 1 hour at room temperature and then washed again with TBST. For detection, an enhanced chemiluminescence-Western blot system (Amersham, Piscataway, USA) was used. For quantification, the X-ray films were put through a scanner (Samsung, Seoul, Korea) and analyzed with LAS image analysis system (Fujifilm, Tokyo, Japan). The picture were analysed with software ImageJ (National institutes of Health, USA) to obtain gray value of each band. Then relative gray value of each marker(gray value of special marker / gray value of β-actin)were calculated and analysed.
4
Osteocyte Apoptosis and Autophagy Analysis
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The protein samples were extracted from osteocytes under Dex treatment with or without rhPTH(1–34) for 72 h. Protein samples were fractionated by SDS-PAGE and electrophoretically transferred onto a nitrocellulose membrane. After blocking, the membrane was incubated overnight in 1 of the following primary antibodies: cleaved caspase 3 (1: 1000), Cleaved-PARP (1: 1000), Beclin-1 (1: 1000), or LC3 (1: 1000), and β-actin (1: 1000) was used as an internal control (Cell signaling technology, Beverly, MA, USA). After 2-h incubation with horseradish peroxidase-conjugated secondary antibody (1: 5000), the images were detected using the Enhanced Chemiluminescence Western Blot System (Amersham Biosciences).
5
Protein Analysis by SDS-PAGE and Western Blot
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Protein samples were analyzed by SDS-PAGE on a 12% gel followed by Western blot analysis using an enhanced chemiluminescence Western blot system (Amersham Biosciences, Piscataway, NJ, USA). An anti-His-tag antibody (1:10,000 [v/v]) (Abcam, Cambridge, UK) was used as a primary antibody, and a horseradish-peroxidase-conjugated anti-rabbit IgG antibody (1:5000 [v/v]) was used as a secondary antibody. Exposure and detection procedures were performed according to the manufacturer’s instructions.
6
Western Blot Analysis of Inflammatory Cytokines
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Between 60 and 100 µg of protein from the nuclear extract, cytoplasmic lysate, or whole cell pellets were subjected to SDS-PAGE (6-12% polyacrylamide gels). Separated proteins were blot transferred onto a nitrocellulose membrane. After blocking with 0.1% Tween 20 and 5% nonfat dry milk in Tris-buffered saline at room temperature for 1 h, the membrane was incubated overnight at 4°C in one of the following primary antibodies: IL-1α (Santa Cruz, CA, USA), IL-1β (Abcam, MA, USA), IL-6 (Abcam, MA, USA) and βactin (Santa Cruz, CA, USA) as an internal control. The membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:2000) for 1 h and detected using the Enhanced Chemiluminescence Western blot System (Amersham Biosciences).
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