Samples were resolved by SDS-PAGE using Criterion™ TGX™ 4–20% precast gel (Bio-Rad) under reducing conditions and transferred to PVDF membranes (Immobilon P). The membranes were blocked with 5% milk in 0.1% Tween-20 and probed with the indicated antibodies and detected using an enhanced chemiluminescent detection system (Millipore). APOB antibody (Millipore, AB742) was used at 1:1,000. Purified mouse LDL was used as positive control (see full scan Western blot images in supplemental data).
Enhanced chemiluminescent detection system
Manufactured by Merck Group
Sourced in United Kingdom
The Enhanced Chemiluminescent Detection System is a laboratory equipment designed for the detection and quantification of proteins in Western blot analysis. It utilizes a chemiluminescent reaction to generate a light signal proportional to the amount of target protein present in the sample. The system includes reagents and consumables necessary for the chemiluminescent detection process.
Automatically generated - may contain errors
Lab products found in correlation
G box ichemi chemiluminescence image capture system, by Syngene (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Horseradish peroxidase, by Proteintech (3 mentions)
Ripa lysis buffer, by Active Motif (2 mentions)
Phosphatase inhibitor, by Active Motif (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Gapdh, by Proteintech (2 mentions)
Phosphatase inhibitor, by Roche (2 mentions)
Criterion tgx 4 20 precast gel, by Bio-Rad (1 mentions)
Anti trx1, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Anti trx2, by Santa Cruz Biotechnology (1 mentions)
Anti bmal1, by Abcam (1 mentions)
Anti t akt, by Cell Signaling Technology (1 mentions)
Anti p akt ser473, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Phosphorylated erk1 2 at thr202 tyr204, by Cell Signaling Technology (1 mentions)
Phosphorylated jnk at thr183 tyr185, by Cell Signaling Technology (1 mentions)
Cox 2, by Santa Cruz Biotechnology (1 mentions)
9 protocols using enhanced chemiluminescent detection system
1
Western Blot Analysis of APOB
2
Western Blot Analysis of Redox Proteins
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Equal amounts of protein sample (15–35 ug/lane) were separated on 12% gels by SDS-PAGE and transferred to PVDF membrane. Membranes were blocked in 5% milk (Tris-buffered saline with 0.2% Tween-20, TBST) for 1 h at room temperature. Primary antibodies were diluted in 5% milk/TBST and incubated for 1 h at room temperature. Anti-TR1 (1:2000), anti-Trx1 (1:2000), anti-Trx2 (1:2000) were purchased from Santa Cruz Biotechnology (Dallas, TX). Anti-TR2 (1:2000), anti-PRX3 (1:2000), anti-PRX2 (1:2000), and anti-PRX-SO2/SO3H (1:2000) were purchased from ABFrontier (Seoul, Korea). Anti-actin antibody (1:5000) was from Millipore (Billerica, MA). The membranes were washed for 5 min with fresh TBST and washed in this manner 5×, after which the membranes were incubated with appropriate species of horseradish peroxidase conjugated secondary antibody (Millipore) diluted in 5% milk/TBST for 1 h at room temperature. The membranes were again washed 5× (5 min each) with TBST and protein bands were visualized with Enhanced Chemiluminescent™ detection system (Millipore).
3
Western Blot Analysis of Circadian Proteins
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Approximately 40 μg of protein was separated on a 10% SDS gel, following which it was transferred to a nitrocellulose membrane. The nonspecific binding sites of the nitrocellulose membrane were blocked using 5% nonfat milk and then incubated with anti-BMAL1 (Abcam) (1:1000), anti-T-AKT (Cell Signaling Technology, Danvers, MA, USA) (1:1000), anti-P-AKT (Ser473) (Cell Signaling Technology) (1:1000), and anti-GAPDH (Abcam) (1:5000) antibodies at 4 °C overnight. After washing, the blot was incubated and diluted with the corresponding peroxidase-conjugated secondary antibodies for 1 h at room temperature. Finally, the protein signals were detected using the enhanced chemiluminescent detection system (Millipore, Billerica, MA), and the ratio of a target protein to that of the intensity of GAPDH was obtained as each target protein level.
4
Western Blot Analysis of Signaling Proteins
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After treatments, the cells were lysed in ice-cold radio immune-precipitation assay (RIPA) lysis buffer (Active Motif, Carlsbad, CA) containing a protease inhibitor cocktail (Sigma) and a phosphatase inhibitor (Active Motif). After determination of protein concentration, the extracted protein was analyzed with western blotting following a standard protocol as described previously28 (link). The primary antibodies against p65 (1:1000, Cell Signaling, Danvers, MA), phosphorylated p65 at Ser 536 (1:1000, Cell Signaling), p38 (1:500, Cell Signaling), phosphorylated p38 at Thr180/Tyr182 (1:200, Cell Signaling), ERK1/2 (1:200, Cell signaling), phosphorylated ERK1/2 at Thr202/Tyr204 (1:200, Cell signaling), JNK (1:1000, Cell signaling), phosphorylated JNK at Thr183/Tyr185 (1:1000, Cell signaling), IκBα (1:200, Santa Cruz, Santa Cruz, CA) and COX-2 (1:200, Santa Cruz) were used to probe the respective target proteins, which were followed by incubations with secondary antibodies and an enhanced chemiluminescent detection system (Millipore, Billerica, MA). Internal loading controls were probed with a GAPDH antibody (1:10000, Proteintech).
5
Western Blot Analysis of Protein Targets
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Total proteins were extracted using ice-cold radioimmunoprecipitation assay lysis buffer (Shenggong, Shanghai, China), supplemented with protease and phosphatase inhibitors (Roche, Basel, Switzerland). Equal amounts of protein were denatured at 100°C for 10 min and then electrophoresed in 10% SDS-polyacrylamide gels. Afterward, blots were wet-transferred to nitrocellulose membranes (Millipore, Billerica, MA). After blocking, blots were incubated with primary antibodies against MT1 (Abcam, Cambridge, United Kingdom), AR, and aromatase (both from Santa Cruz Biotechnology, United States) at 1:600–1:1000 dilutions and then incubated against secondary antibodies. An enhanced chemiluminescent detection system (Millipore) was used to detect bands with peroxidase activity. The same blot was probed with GAPDH (Proteintech, Chicago, United States) as the internal loading control. The bands were visualized using a G-Box iChemi Chemiluminescence Image Capture System (Syngene, Frederick, MD, United States).
6
Western Blot Analysis of MAPK Signaling
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Total protein was extracted using ice-cold radio-immunoprecipitation assay lysis buffer (Cwbio) containing a phosphatase inhibitor and a protease inhibitor cocktail (both from Roche). Protein from each sample was electro-phoresed in a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and then transferred onto a nitrocellulose blot. After 1 hour of blocking with 5% nonfat milk, the blot was incubated overnight at 4°C with antibodies against phospho-JNK (Thr183/Tyr185; 1:1000), and total JNK (1:1000), phospho-p38 MAPK (Thr180/Tyr182; 1:1000), total p38 MAPK (1:1000). On the second day, the blot was washed and then incubated with the respective secondary antibody conjugated to horseradish peroxidase (1:1500) for 1 hour. An enhanced chemiluminescent detection system (Millipore) was used to detect the bands with peroxidase activity. A G-Box iChemi Chemiluminescence image capture system (Syngene) was used to visualize the bands. The same blot was also probed with α-tubulin (1:1000) as internal controls. All antibodies were from Cell Signaling Technology (CST, Massachusetts, USA).
7
Protein Extraction and Western Blot Analysis
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Total cellular protein was extracted from the above treated cells using an ice-cold radio immunoprecipitation assay (RIPA) lysis buffer (Active Motif) containing a protease inhibitor cocktail (Roche) and a phosphatase inhibitor (Roche). Tween/Tris-buffered saline solution, the membrane was incubated with appropriate secondary antibodies conjugated with horseradish peroxidase (1:5000; Proteintech) for 1 h. The enhanced chemiluminescent detection system (Millipore) was used to detect the bands with peroxidase activity. To control sampling error, the same blot was probed for α-tubulin (1:1000; Proteintech)
for internal loading control. The bands were visualized using a G-Box iChemi Chemiluminescence Image Capture system (Syngene). The abundance of phosphorylated STAT3 and SRC was expressed as the ratio of their band densities over total STAT3 or SRC respectively.
for internal loading control. The bands were visualized using a G-Box iChemi Chemiluminescence Image Capture system (Syngene). The abundance of phosphorylated STAT3 and SRC was expressed as the ratio of their band densities over total STAT3 or SRC respectively.
8
Quantitative Western Blot Analysis
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Whole cell lysate protein was prepared from the above treated cells by using ice-cold radio immunoprecipitation assay buffer (Active Motif, Carlsbad, CA) containing a complete protease inhibitor cocktail (Roche, Basel, Schweiz) and phosphatase inhibitor (Active Motif). A standard procedure of western blotting was performed as described previously27 (link). Briefly, after determination of protein concentration with Bradford assay, 20 µg protein of each sample was electrophoresed in SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Merck Millipore). After blocking and incubation with primary antibodies (Table 1 ), the membranes were incubated with appropriate secondary antibodies conjugated with horseradish peroxidase (1:5000; Proteintech). The peroxidase activities of target protein bands were detected using an enhanced chemiluminescent detection system (Merck Millipore) and visualized by using a G-Box capture system (Syngene, Cambridge, UK). The abundance of phosphorylated PDHE1α was expressed as the ratio over total PDHE1α. The abundance of PDK4 and 11β-HSD2 was expressed as the ratio over the housekeeping gene α-Tubulin.
9
Profiling mTOR and AKT Signaling Cascades
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Total protein was extracted using ice cold radioimmunoprecipitation assay (RIPA) lysis buffer (Cwbio), containing a phosphatase inhibitor (Roche) and a protease inhibitor cocktail (Roche). Then, 25 µg of protein from each sample was electrophoresed in SDS polyacrylamide gels and then transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with antibodies against SGK3 (serum/ glucocorticoid-regulated kinase family member 3; 1:500 dilution; Proteintech), mTOR (mammalian target of rapamycin kinase; 1:1000 dilution; Cell Signaling), Phospho-mTOR (1:1000 dilution; Ser2448, Cell Signaling), AKT (protein kinase B; 1:1000 dilution; Cell Signaling), Phospho-AKT (1:1000 dilution; Ser473, Cell Signaling) overnight at 4°C. After washing four times with Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated with the appropriated secondary antibody, conjugated with horseradish peroxidase (Proteintech) for 1 h at room temperature. Bands with peroxidase activity were detected by an enhanced chemiluminescent detection system (Merck Millipore) and visualized using a G-Box chemiluminescence image capture system (Syngene). GAPDH (glyceraldehyde-3-phosphate dehydrogenase; 1:5000 dilution; Proteintech) as a protein loading control.
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