Lung tissues were homogenized in 0.25 mol/L sucrose buffer, whereas cells were lysed with cell lysis buffer containing protease inhibitor (Sigma, St. Louis, MO; P8340) and phosphatase inhibitor cocktails (Sigma; P5726). For tissue samples, the homogenates were centrifuged at 3000 × g, 4°C, for 10 minutes, followed by a centrifugation at 100,000 × g for 60 minutes. Protein concentrations were measured using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Protein (50 μg) was resolved onto a 10% SDS-PAGE gel and electrophoretically transferred onto polyvinylidene difluoride membranes, which were probed with the following antibodies: PAI-1 (Molecular Innovation, Novi, MI; ASMPAI-GF), p53 (Santa Cruz Biotechnology, Dallas, TX; SC-6243), p21 (Santa Cruz Biotechnology; SC-397), procollagen 1α1 (Santa Cruz Biotechnology; SC-8784-R), fibronectin (BD Biosciences; 610077), and β-actin (Sigma; A5441; protein loading control), and then with the corresponding horseradish peroxidase–conjugated secondary antibodies. The protein bands were visualized using the electrochemiluminescence detection system (Amersham, Piscataway, NY), semiquantified using ImageJ version 1.53c software (NIH, Bethesda, MD: https://imagej.nih.gov/ij , last accessed October 29, 2020), and normalized by β-actin.
Electrochemiluminescence detection system
Manufactured by Cytiva
Sourced in United States
The Electrochemiluminescence detection system is a versatile laboratory instrument designed to measure and analyze electrochemiluminescent signals. It provides a reliable and sensitive platform for various applications that require the detection and quantification of electrochemiluminescent analytes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Fibronectin, by BD (1 mentions)
Procollagen 1α1, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ripa buffer, by Santa Cruz Biotechnology (1 mentions)
Colored markers, by Bio-Rad (1 mentions)
Rabbit anti sr b1, by Novus Biologicals (1 mentions)
Proliferating cell nuclear antigen (pcna), by Abcam (1 mentions)
Fresh extraction buffer, by Merck Group (1 mentions)
Protease inhibitor and phosphatase inhibitor, by GoldBio (1 mentions)
Goat anti mouse igg, by R&D Systems (1 mentions)
Cleaved parp, by Affinity Biosciences (1 mentions)
Cytochrome c, by Affinity Biosciences (1 mentions)
Cleaved caspase 3, by Affinity Biosciences (1 mentions)
Bcl2 associated x (bax), by Affinity Biosciences (1 mentions)
7 protocols using electrochemiluminescence detection system
1
Protein Analysis of Lung Tissues
2
Protein Expression Analysis via SDS-PAGE and Immunoblotting
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SDS-PAGE and immunoblotting were performed according to standard procedures. The protein samples were loaded onto 10% or 15% SDS-PAGE for electrophoresis and blotting. The membranes were incubated overnight at 4 ℃ with primary antibodies. After washing, the membranes were incubated in a secondary IgG antibody conjugated to horseradish peroxidase for 1 h, and the protein levels were detected using an electro-chemiluminescence detection system (Amersham Pharmacia Biotech, Uppsala, Sweden). Densitometric values were quantified using Quantity One software (Bio-Rad, Hercules, CA, USA).
3
Western Blot Analysis of Cellular Proteins
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After the indicated cell treatment(s), the cells were harvested, and whole-cell lysates were prepared using RIPA buffer (Santa Cruz) [11 , 18 ]. Equal aliquots of whole-cell protein (either 100 μg unprecipitated whole-cell lysate or one half of the precipitated protein from 500-μg whole-cell lysate) were electrophoresed on 4-12% SDS-polyacrylamide gradient gels, transferred to nitrocellulose membranes (Millipore, Bedford, MA), and blotted using primary antibodies against BRCA1 (C-20, rabbit polyclonal, 1:200 dilution; Santa Cruz), cathepsin D (R-20, goat polyclonal, 1:300; Santa Cruz), ER (F10, mouse monoclonal, 1:500; Santa Cruz), or actin (goat polyclonal, sc-1615, 1:400; Santa Cruz). The membranes were blotted with the appropriate secondary antibodies (1:1000; Santa Cruz), and the blotted proteins were visualized using an electrochemiluminescence detection system (Amersham Biosciences), with colored markers (Bio-Rad Laboratories, Hercules, CA) as molecular size standards.
4
Immunoblot Analysis of SR-B1 Protein
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Immunoblot analysis was performed as described previously [1 (link)]. Briefly, 4 x105 cells of each cell lines seeded in 6 well plates and cells were grown until confluent. Equal amounts of proteins were separated by 10% SDS-PAGE under reducing conditions and transferred to a polyvinylidene difluoride membrane. The primary antibody used was Rabbit anti- SR-B1 (Novus Biologicals, Littleton, USA). Immunoreactivity was detected with the electrochemiluminescence detection system (Amersham Biosciences, Buckinghamshire, UK). Anti-human β-actin antibody was included to normalize against unequal loading.
5
Western Blot Analysis of Apoptosis Markers
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Grafts and SPCs were lysed in fresh extraction buffer (Sigma-Aldrich, St. Louis, MO, USA) supplemented with a protease inhibitor and phosphatase inhibitor (Gold Biotechnology, St. Louis, MO, USA). The extracted proteins were separated on a 15% SDS-polyacrylamide gel and electroblotted onto a polyvinylidene fluoride membrane. The membrane was blocked using 5% skim milk-containing TBST for 60 min at 20–25°C and incubated with primary antibodies against Bcl-2 (Affinity), Bax (Affinity), cytochrome c (Affinity), cleaved-caspase-3(Affinity), cleaved-PARP (Affinity), and PCNA (Abcam) overnight at 4℃. The membranes were then washed with TBST and incubated with goat anti-mouse IgG (R&D Systems, Inc., Minneapolis, MN, USA) and goat anti-rabbit IgG (R&D Systems) secondary antibodies. Bound antibodies were detected using an electrochemiluminescence detection system (Amersham Life Science, Arlington Heights, IL, USA). β-actin was used as the loading control.
6
Protein Expression Analysis by Western Blot
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Total protein content was determined by a BCA Protein Assay Kit (Beyotime) after protein extraction using RIPA lysis buffer. Then, 25 μg of protein extracted from each sample were resolved by 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, CA, USA) by electroblotting. The blotted membranes were blocked with 5% non-fat milk for 1-2 h at RT and then were probed with anti-Nampt (1:1000 dilution, BETHYL, USA) and anti-β-actin (1:1500 dilution, Abcam, UK) diluted in tris-buffered saline (TBS) overnight at 4 °C. After incubating with horseradish peroxidase-conjugated with anti-rabbit IgG secondary antibody (1:2000 dilution, Proteintech, USA), protein blots were visualized using an enhanced Electro-Chemi-Luminescence detection system (Amersham Biosciences, Piscataway, NJ, USA).
7
Western Blot Analysis of Scd2 Protein
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After protein extraction using RIPA lysis buffer, the total protein content was determined using the BCA protein assay kit (Beyotime). Then, 30 μg of protein extracted from each sample was resolved by 10% SDS-PAGE gels and transferred by electroblotting onto PVDF membranes (Millipore, Billerica, CA, USA). The blotted membranes were incubated with 5% skim milk for 1–2 h at RT and then detected overnight at 4°C with anti-Scd2 (1:500 dilution, Santa Cruz, USA) and anti-β-actin (1:2000 dilution, Proteintech, USA) diluted in TBST. Incubation with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2000 dilution, Beyotime Biotechnology, China) was performed for 1–2 h followed by washing with TBST. Finally, protein blots were visualized using an enhanced Electro-Chemi-Luminescence detection system (Amersham Biosciences, Inc., Piscataway, NJ, USA). β-actin was used as an internal standard.
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