HUVECs were treated with or without estradiol at a fixed time after preincubation with fixed concentrations. Cells were harvested and lysed by scraping in ice-cold RIPA buffer (Santa Cruz, CA, USA). Protein concentrations were determined by BCA assay (Solarbio, Beijing, China), and total protein for each 50 μg sample was resolved on polyacrylamide gel and blotted onto PVDF membranes for immunoblotting. The membranes were incubated with 5% nonfat dry milk in TBST for 1 h at room temperature. The primary antibodies were incubated with the membranes, including anti-FAK (1:1000), anti-FLNA (1:250000), anti-ZYX (1:10000), anti-BCAR1 (1:1000), anti-SHC1 (1:5000) and anti-GAPDH (1:5000) (Abcam, Cambridge, MA, USA), and then incubated at 4°C overnight. After incubation, the membranes were washed, incubated with horseradish-peroxidase secondary antibody IgG (1:5000) for 1 h, and washed again. Protein band densities were quantified using an enhanced chemiluminescence detection system (syngene, USA), and the protein levels were analyzed using ImageJ software.
Enhanced chemiluminescence detection system
Manufactured by Syngene
Sourced in United Kingdom
The Enhanced Chemiluminescence Detection System is a laboratory equipment used to detect and quantify proteins and other biomolecules in various applications, such as Western blotting. The system utilizes chemiluminescent substrates that emit light upon reaction with the target molecules, which is then detected and measured by a sensitive camera or imaging device.
Automatically generated - may contain errors
Lab products found in correlation
Bca assay, by Solarbio (1 mentions)
Anti flna, by Abcam (1 mentions)
Anti shc1, by Abcam (1 mentions)
Anti fak, by Abcam (1 mentions)
Ripa buffer, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Hrp linked secondary antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Bca assay kit, by Takara Bio (1 mentions)
4 protocols using enhanced chemiluminescence detection system
1
Estradiol Effect on Focal Adhesion Proteins
2
Protein Expression Analysis Protocol
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Proteins are obtained by lysing cells with radio immunprecipitation assay lysis buffer containing 1% phenylmethylsulfonyl fluoride. Then the protein is quantified using a BCA protein assay kit, separated using SDS-PAGE, and shifted to NC membrane. The membrane was incubated with milk, primary antibody and secondary antibody in order. Finally, using an enhanced chemiluminescence detection system (Syngene, Cambridge, UK) to evaluate the level of protein expression.
3
Protein Expression Quantification in HT1080 Cells
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HT1080 cells were incubated with EAA (0.1, 1, and 10 μM) and PMA (10 ng/mL) in a CO2 incubator for 24 h by referring to the method of Chen et al. [38 (link)]. Proteins were obtained by lysing cells using RIPA buffer containing 1% of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Separation was performed using SDS-PAGE. The proteins were then transferred onto nitrocellulose (NC) filter membranes. Primary antibodies were incubated overnight at 4 °C (1:1000), followed by sufficient incubation of secondary antibodies for 2 h (1:2000). Finally, the membranes were photographed using an enhanced chemiluminescence detection system (Syngene, Cambridge, UK), and the images were analyzed using ImageJ software.
4
Western Blot Protein Analysis Protocol
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The total protein from GC cells were extracted using radioimmune precipitation (RIPA) buffer (Wolsen Biotech,) supplemented with phosphatase and protease inhibitors as well as phenylmethyl sulfonyl (PMSF) (Sigma). A BCA assay kit (Takara Bio, Inc.) was then used for quantifying protein concentration. After that, prepared samples were isolated via SDS‐PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, CA). Ten per cent skim milk was then used for blocking for 90 min, followed by incubating overnight at 4°C using appropriate primary antibodies. The membranes were treated with HRP‐linked secondary antibodies for 1.5 h on the second day (Cell Signalling Technology, Inc.). The membranes were cleaned 3 times with Tris‐buffered saline‐Tween 20 after each incubation step. At the end, the membranes were observed using an enhanced chemiluminescence detection system (Syngene Europe) and analysed with ImageJ software (National Institutes of Health). For primary antibodies used in this study, see Table 3 .
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