The cells were treated with kaempferol for 24 h, harvested, and lysed in protein extraction solution (Intron Biotechnology, Inc., Gyeonggi, Korea) containing protease inhibitors and phosphatase inhibitors for 10 min at 4°C. The total protein concentration in the supernatant was measured by using the Bradford assay. After heating at 95°C for 5 min, the samples of total protein (40 μg) were subjected to 6~15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) by the application of 100 V for 60~100 min. The membranes were incubated with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) with 0.05% Tween 20 (TBST) for 30 min at room temperature and then with primary antibodies diluted 1: 200~1:1,000 in 5% BSA in TBST overnight at 4°C. The membranes were washed three times with TBST and incubated with the appropriate secondary antibodies. The protein bands were detected by using an enhanced chemiluminescence detection kit (Intron Biotechnology, Inc.) and an LAS-1000 Imager (Fuji Film Corp., Tokyo, Japan).
Enhanced chemiluminescence detection kit
Manufactured by iNtRON Biotechnology
Sourced in Japan
The Enhanced Chemiluminescence Detection Kit is a laboratory instrument designed to detect and quantify proteins in biological samples. The kit utilizes chemiluminescent-based detection methods to enable sensitive and accurate protein analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protein extraction solution, by iNtRON Biotechnology (4 mentions)
Las 1000 imager, by Fujifilm (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Imagequant las 4000 imager, by Fujifilm (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Nitrocellulose blotting membrane, by Bio-Rad (1 mentions)
Anti nrf2 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti keap1 antibody, by Santa Cruz Biotechnology (1 mentions)
Bca assay kit, by iNtRON Biotechnology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Traf6, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Pall Corporation (1 mentions)
Imagequant las 500, by GE Healthcare (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
10 protocols using enhanced chemiluminescence detection kit
1
Western Blot Analysis of Kaempferol-Treated Cells
2
Nrf2 and Keap1 Protein Expression
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A549 cells were lysed by protein extraction solution about 70 μL (iNtron Biotechnology, Seoungnam, Korea). The protein concentrations of samples were determined through the bicinchoninic acid (BCA) assay kit (iNtron Biotechnology, Korea). A 30 μg of protein were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were electrophoresed for 5 h (60 mA) at room temperature and then transferred onto a nitrocellulose blotting membrane (Bio-rad, Hercules, CA, USA) for overnight. The membrane was blocked with 5% skim milk in tris-buffered saline (TBS) with 0.01% Tween-20 for 1 h and incubated with rabbit polyclonal anti-Nrf2 antibody (1:250 dilution, Santa cruz, sc-13032, Dallas, TX, USA) or goat polyclonal anti-keap1 antibody (1:300 dilution Santa cruz, sc-15246, USA) for overnight. To detect Nrf2, keap1, Horseradish peroxidase-conjugated anti-rabbit IgG or anti-goat IgG (both at 1:5,000 dilution, KPL, Gaitherburg, MD, USA) were incubated as a secondary antibody for 1 h at room temperature. Immunoreactivity was visualized by an enhanced chemiluminescence detection kit (iNtron Biotechnology, Korea).
3
Western Blot Analysis of Endothelial Cells
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ECs were treated with AS for 24 h, and Western blot was performed according to a previously described method [25 (link)]. Briefly, ECs were harvested and lysed in protein-extraction solution (Intron Biotechnology, Inc., Kyunggi, Korea) containing protease inhibitors and phosphatase inhibitors for 10 min at 4 °C. The total protein concentration in the supernatants was measured using the Bradford assay [11 (link)]. After heating at 95 °C for 5 min, total protein samples (30 μg) were subjected to 6% to 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) at 100 V for 60 min to 100 min. The membranes were incubated with 5% bovine serum albumin in Tris-buffered saline with 0.05% Tween 20 (TBST) for 30 min at room temperature, and then with diluted primary antibodies (1:200–1:1000) in 5% BSA in TBST overnight at 4 °C. The membranes were washed three times with TBST and incubated with the corresponding secondary antibodies. Protein bands were detected using an enhanced chemiluminescence detection kit (Intron Biotechnology, Inc.) and a LAS-1000 Imager (Fuji Film Corp., Tokyo, Japan). Semi-quantitative analysis of densitometry results was performed using Image J software (National Institutes of Health, Bethesda, MD, USA).
4
Western Blot Protein Analysis Protocol
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Total cell lysates were mixed with lysis buffer, and boiled for 10-20 min at 100°C. The protein concentrations of the cell lysates were determined by the BCA protein assay (Byun et al., 2019 (link)). Samples containing equal amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis. The separated proteins were electrically transferred to PVDF membranes (Millipore, Darmstadt, Germany) activated with 100% methanol. The membranes were blocked using 5% bovine serum albumin (BSA, Sigma-Aldrich) in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 30 min at least at room temperature. The desired primary antibodies were diluted in 2.5% BSA (1:500-1:10,000) overnight at 4°C. The membranes were washed three times with PBST and were then incubated with the appropriate secondary antibodies diluted in TBST for 2 h at room temperature. The membranes were washed three times with PBST, and then were visualized using an enhanced chemiluminescence detection kit (Intron Biotechnology, Sungnam, Korea). The membranes were analyzed using an ImageQuant LAS 4000 imager (Fujifilm Corp., Tokyo, Japan).
5
Western Blot Analysis of Hesperetin-Treated Cell Lysates
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Cells were treated with hesperetin for 24 h. Harvested cells were lysed in protein extraction solution (Intron Biotechnology, Inc., Seongnam, Korea) containing protease inhibitors and phosphatase inhibitors for 10 min at 4°C. The total protein concentration in the supernatants was measured by the Bradford assay. After heating at 95°C for 5 min, total protein samples (40 μg) were subjected to 6~15% SDS-PAGE. The proteins were transferred onto PVDF membranes (Millipore Corporation, Bedford, MA) at 100 V for 60~100 min. The membranes were incubated with 5% BSA in TBST (TBS with 0.05% Tween 20) for 30 min at room temperature and then with primary antibodies diluted (1:200~1:1,000) in 5% BSA in TBST overnight at 4°C. The membranes were washed three times with TBST and incubated with the corresponding secondary antibodies. Protein bands were detected using an enhanced chemiluminescence detection kit (Intron Biotechnology, Inc.) and an LAS-1000 Imager (Fuji Film Corp., Tokyo, Japan).
6
Rhododendrin Inhibits IMQ-Induced Keratinocyte Signaling
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Normal human epidermal keratinocyte cells were pre-treated with rhododendrin at various doses for 1 h and subsequently stimulated with 1 μg ml−1 IMQ for the indicated times. The cells were harvested, and the collected cell pellets were lysed for 30 min at 4 °C in ice-cold RIPA buffer containing a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Samples containing equal amounts of protein were resolved by SDS–polyacrylamide gel electrophoresis and analyzed by immunoblotting. Briefly, membranes containing the transferred polypeptides were blocked in buffer containing 5% skim milk in Tris-buffered saline (pH 7.4) and 0.1% Tween 20 (TBST) for 1 h at room temperature and subsequently probed with primary antibodies against the indicated target molecules at 4 °C overnight. The membranes were then probed with horseradish peroxidase-conjugated secondary antibodies (Invitrogen, Grand Island, NY, USA) and processed using an enhanced chemiluminescence detection kit ((iNtRON Biotechnology, Gyeonggi-do, Republic of Korea). The signals were detected on X-ray film developed in a darkroom or with an Image-Analyzer System (LAS-3000, Fujifilm, Tokyo, Japan). TRAF6 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and immunoprecipitation was performed as previously described.21 (link)
7
Western Blotting Analysis of iNOS and COX-2
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The Western blotting analysis was performed as previously described [31 (link)]. Briefly, the samples with cell lysates were boiled for 10 min at 100 °C. The concentrations of proteins in the cell lysates were quantified using the bicinchoninic acid method. Equal amounts of protein were subjected to 8%–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) activated with 100% methanol. The membranes were blocked using 5% BSA in a mixture of Tris-buffered saline and Tween 20 (1×), and subsequently probed with the following antibodies: anti-iNOS, COX-2, and GAPDH (Cell Signaling Technology, Beverly, MA, USA). The blots were visualized using an enhanced chemiluminescence detection kit (Intron Biotechnology, Sungnam, Korea) and an ImageQuant LAS-4000 Imager (Fujifilm Corp., Tokyo, Japan).
8
Western Blot Protein Analysis
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Samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Pall Corp., Port Washington, NY, USA). Membranes were blocked for 1 hour in a blocking buffer (5% skim milk in 25 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 0.1% Tween 20) and incubated overnight at 4℃ with specific primary antibodies for target signaling. Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 hours at room temperature, and the signals were detected by using radiography after reaction with an enhanced chemiluminescence detection kit (iNtRON Biotechnology, Seongnam, Korea).
9
Western Blot Analysis of Myricetin-Treated Cells
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The cells were treated with myricetin for 24 hours. Harvested cells were lysed in protein extraction solution (Intron Biotechnology, Inc., Seongnam, Korea) containing protease inhibitors and phosphatase inhibitors for 10 minutes at 4°C. The total protein concentration in the supernatant was measured by using the Bradford assay. Forty microgram samples of the total proteins were heated at 95°C for 5 minutes and subjected to 6% to 15% SDS-PAGE. The proteins were transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) at 100 V for 60 to 100 minutes. The membranes were incubated with 5% bovine serum albumin (BSA) in TBS with 0.05% tween 20 (TBST) for 30 minutes at room temperature and then with primary antibodies diluted at dilutions of 1 : 200 to 1 : 1,000 in 5% BSA in TBST overnight at 4°C. The membranes were washed three times with TBST and incubated with the corresponding secondary antibodies. The protein bands were detected by using an enhanced chemiluminescence detection kit (Intron Biotechnology, Inc.) and an LAS-1000 Imager (Fuji Film Corp., Tokyo, Japan).
10
Western Blot Protein Quantification
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Protein concentrations were determined by DC Protein Assay (Bio-RAD, Hercules, CA, USA). The protein extracts were separated on 8~15% SDS-PAGE, and transferred onto polyvinylidene fluoride membranes. The membranes were saturated with 3% skim milk, incubated overnight at 4 °C with primary antibodies (1:1000), washed, and then incubated for 2 h with secondary antibodies, i.e., peroxidase-conjugated goat anti-mouse, anti-goat or anti-rabbit immunoglobulin G (Santa Cruz Biotechnology, 1:5000 to 1:10,000). Signals were revealed by autoradiography with the enhanced chemiluminescence detection kit (iNtRON Biotechnology, Korea) using ImageQuant LAS 500 (GE Healthcare, Uppsala, Sweden). Western bands were determined by densitometry of bands using Image J software.
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