Ginkgetin and GSK1016790A (GSK101) were purchased from Sigma-Aldrich (St. Louis, MO), and FLIPR calcium 6 assay kit was obtained from Molecular Device (Sunnyvale, CA). Human native LDL (nLDL) was purchased from Stemcell Technologies (Vancouver, Canada). We incubated nLDL with 5 µM CuSO4 for 12 h at 37 °C to prepare copper-oxidized LDL (oxLDL)7 (link),14 (link),16 . Fluorescent dye-labeled, DiI-oxLDL, was purchased from Invitrogen (Carlsbad, CA), and MCSF from R&D (Minneapolis, MN). Antibodies for FACS analysis were: TRPV4 (Millipore; Burlington, MA), CD36 (R&D; Minneapolis, MN), TLR4 and TLR6 (Novus Biologicals; Centennial, CO). PE-conjugated secondary antibodies were from R&D (Minneapolis, MN), and PE-conjugated isotype controls were from BD (Franklin Lake, NJ). For quantitative polymerase chain reaction (qRT-PCR), we used RNeasy kit from QIAGEN (Redwood City, CA). All primers were purchased from Bio-Rad (Hercules, CA). Cell culture media, cell culture related products, and western blot reagents were obtained from Thermo Fischer Scientific (Waltham, MA).
Western blot reagent
Manufactured by Thermo Fisher Scientific
Sourced in Netherlands
Western blot reagents are a set of laboratory chemicals and materials used to perform the Western blot technique. This technique is used to detect and quantify specific proteins in a complex sample. The reagents include solutions for protein extraction, separation, transfer, and detection, allowing researchers to analyze protein expression and modifications.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Cell culture media, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Ginkgetin, by Merck Group (1 mentions)
Gsk1016790a gsk101, by Merck Group (1 mentions)
Flipr calcium 6 assay kit, by Molecular Devices (1 mentions)
Dii oxldl, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
M csf, by R&D Systems (1 mentions)
Kbr disks, by Bruker (1 mentions)
Facs canto 2, by Bio-Rad (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Annexin 5, by Thermo Fisher Scientific (1 mentions)
Accutase enzyme, by Thermo Fisher Scientific (1 mentions)
Rna isolation and cdna synthesis kits, by Thermo Fisher Scientific (1 mentions)
Cd ch3coo 2 2h2o, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
6 protocols using western blot reagent
1
Copper-Oxidized LDL Preparation and Analysis
2
Synthesis and Characterization of Platinum-based Anticancer Compounds
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Cd(CH3COO)2.2H2O and 5-aminotetrazole, cisplatin (cis-diamminedichloroplatinum(II)) were obtained from Sigma Aldrich, CA, USA. Culturing medium DMEMTM and phenol red free medium DMEM/F12, PBS, FBS, FCS, Trypsin, Accutase enzyme, Penicillin, Presto- Blue27 , Annexin V, propidium iodide (PI) solutions, RNA isolation and cDNA synthesis kits, primers, antibodies and Western blot reagents were obtained from Thermo Fisher Scientific and Gibco/Invitrogen, Breda, the Netherlands.
FT-IR spectra were determined on a Bruker Tensor 27 FT-IR spectrophotometer with KBr disks in the range of 4000–400 cm−1 (Columbia, MD, USA). QTOF LC/MS spectrometer was used for mass spectrometry (Billerica, MA, USA). 1HNMR spectroscopy were performed at room temperature on the NMR Bruker Advance 400 MHz magnet (Agilent, USA) using DMSO-d6. Elemental analysis, C H N, was performed by Elemental analyzer Vario EL III (Sydney, Australia). X- ray crystallography was performed on an SuperNova, Dual, Cu at zero, Atlas diffractometer, using Cu Kα radiation λ = 1.54184 Å at 130 K. Facs Canto II (Bio Rad) flow cytometry system (Mexico, USA) with Flow Logic TM program, was used for cell cycle AnnexinV and PI evaluation. Quantitative PCR (qPCR) was performed by Mic qPCR, Bio Molecular system (Santa Clara, USA).
FT-IR spectra were determined on a Bruker Tensor 27 FT-IR spectrophotometer with KBr disks in the range of 4000–400 cm−1 (Columbia, MD, USA). QTOF LC/MS spectrometer was used for mass spectrometry (Billerica, MA, USA). 1HNMR spectroscopy were performed at room temperature on the NMR Bruker Advance 400 MHz magnet (Agilent, USA) using DMSO-d6. Elemental analysis, C H N, was performed by Elemental analyzer Vario EL III (Sydney, Australia). X- ray crystallography was performed on an SuperNova, Dual, Cu at zero, Atlas diffractometer, using Cu Kα radiation λ = 1.54184 Å at 130 K. Facs Canto II (Bio Rad) flow cytometry system (Mexico, USA) with Flow Logic TM program, was used for cell cycle AnnexinV and PI evaluation. Quantitative PCR (qPCR) was performed by Mic qPCR, Bio Molecular system (Santa Clara, USA).
3
Assessing Cellular Stress Responses
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MTT, Cycloheximide (CHX), MG132 and Flag antibody were purchased from Sigma. Antibodies to Sirt3, COX IV, hexokinase II, VDAC1, Mcl-1, survivin and prohibitin were purchased from Cell Signaling Technologies. LC3 antibody was purchased from nanoTools. α-tubulin antibody was purchased from Santa Cruz. Atg12 antibody was purchased MBL. p62 antibody was purchased fromEngo Life Science. All cell culture media were purchased from Invitrogen. Western blot reagents were obtained from Pierce Biotechnology.
4
Apoptosis Signaling Pathway Evaluation
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Amplexicaule A, B (purity = 99.5%) were dissolved in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO) at concentration of 250 mg/ml, and stored at −20°C. Polyclonal antibodies against MCL-1, BCL-2, BAX, cleaved-caspase-3, -8, -9, cleaved-PARP, phospho-Akt, Akt, phospho-mTOR, phospho-P70 S6k and β-actin were purchased from Cell Signaling (Beverly, MA). Horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) was purchased from Abcam Biotechnology (USA). All cell culture media and other reagents were purchased from Invitrogen (Carlsbad, CA). Western blot reagents were obtained from Pierce Biotechnology.
5
Evaluating Myocardial Remodeling Markers
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According to the above-described steps in the subsection titled “Myocardial morphological changes and myocardial cell apoptosis in rats,” quantitative PCR (Thermo Fisher Scientific) and western blot reagent (Invitrogen Inc.) were used to detect myocardial tissue CD147 (antibody 1:1000), the mRNA and protein expression of MMP-9 (antibody 1:1000), and TIMP-1 (antibody 1:1000). Gelatin zymography was used to detect MMP-9 activity (Zymogram Gels; Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, protein was extracted from frozen samples containing 10 mg of protein and mixed with 10 mL of sample buffer, then subjected to SDS-PAGE in 5% polyacrylamide gels that were copolymerized with 2 mg/mL of gelatin at 4°C for 1 hour. After electrophoresis, the gels were washed twice in the rinsing buffer for 1 hour at room temperature to remove SDS. They were then incubated for 36 hours at 37°C in the incubation buffer. The gels were stained with 0.1% Coomassie Brilliant Blue R-250 for 30 minutes and destained for 8 hours in a solution of 10% acetic acid and 10% isopropanol. The proteolytic activity was shown as clear bands against the blue background of stained gelatin. The experiment was repeated three times.
6
Protein Expression Analysis by Western Blot
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After cells handling, the culture medium was removed, and protein lysate (Roche) was added to isolate the total protein. Following gel electrophoresis, 50 µg total protein was transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then sealed with 5% skimmed milk at room temperature (RT) for one hour, cleared with TBST three times (10 min each), and incubated with the primary antibodies (1:1000; Abcam, MA, USA) of Bcl-2 (ab32124), Bax (ab32503), p-PI3K (ab182651), PI3K (ab154598), p-AKT (ab18785), AKT (Ab131168), ITGA11 (ab198826), and β-actin (ab115777) overnight at 4°C. After being flushed with TBST, the membranes were kept at RT with horseradish peroxidase (HRP)-tagged anti-rabbit secondary antibody (1:300) for 1 hour. Subsequently, they were rinsed with TBST three times (10 min each). Finally, the Western blot reagent (Invitrogen) was adopted for color imaging, and the gray intensity was analyzed by Image J.
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