The protein of brain tissues from human and mouse were extracted, and protein concentration was measured using a BCA protein assay. Extracted protein was mixed with 5× loading buffer, electrophoresed in 10% sodium dodecyl sulfate polyacrylamide gel, and transferred onto a polyvinylidene difluoride membrane. The membranes were immersed in 5% skim milk for 2 h at room temperature. After washing with TBST, the membranes were incubated overnight at 4 °C with the primary antibodies. The primary antibodies used in this study were as follows: anti-GAPDH (1:1000, Abcam), anti-ERβ (1:500; from the Jan-Ake Gustafsson's laboratory, Karolinska Institute, Novum, Sweden), anti-VGAT (1:1000, Abcam), anti-VGluT1 (1:1000, Abcam), and anti-Glutamine Synthetase (1:1000, Abcam). After washing, the membranes were incubated with the anti-rabbit or anti-mouse secondary antibodies for 2 h at room temperature. The specific protein bands were visualized in membranes by the chemiluminescence method (Amersham, Piscataway, NJ), and the optical density of protein bands were analyzed in Bio-Rad Image-Lab 6.0 software.
Chemiluminescence method
Manufactured by Cytiva
Sourced in United States, Sweden
Chemiluminescence method is a laboratory technique used to detect and quantify the presence of specific molecules or analytes. The method relies on the emission of light as a result of a chemical reaction. The light produced is then measured and analyzed to determine the concentration or presence of the target substance.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Cytiva (2 mentions)
X omat s films, by Cytiva (2 mentions)
Image lab 6, by Bio-Rad (1 mentions)
Anti glutamine synthetase, by Abcam (1 mentions)
Anti erβ, by Abcam (1 mentions)
Anti vglut1, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
Phospho erk1 2, by Abcam (1 mentions)
Anti flag, by Merck Group (1 mentions)
β catenin, by Proteintech (1 mentions)
β actin, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
N cadherin, by R&D Systems (1 mentions)
Caspase 3, by Santa Cruz Biotechnology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
5 protocols using chemiluminescence method
1
Western Blot Analysis of Brain Proteins
2
Western Blot Analysis of Epithelial-Mesenchymal Transition
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The tissues and cells were lysed with RIPA lysis buffer in addition to 1 mM PMSF. The protein concentration was determined by Coomassie protein assay (Pierce, Rockford, IL, USA). Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, Hercules, CA, USA) and transferred onto a nitrocellulose membrane (Amersham, Chalfont, UK). The membranes were incubated with primary antibodies (aPKC-ι, abcam, Cambridge, UK; P300, abcam, Cambridge, UK; Elk1/phospho-Elk1, abcam, Cambridge, UK; E-cadherin, Proteintech, Rosemont, USA; β-catenin, Proteintech, Rosemont, USA; Vimentin, Proteintech, Rosemont, USA; N-cadherin, Proteintech, Rosemont, USA; Par6, abcam, Cambridge, UK; phospho-NF-kB, abcam, Cambridge, UK; phospho-Erk1/2, abcam, Cambridge, UK; Anti-His, Sigma-Aldrich, St, Louis, USA; Anti-Flag, Sigma-Aldrich, St, Louis, USA) at 4 °C overnight. GAPDH (Proteintech, Rosemont, USA) and β-actin (Proteintech, Rosemont, USA) were used as internal controls. Peroxidase-conjugated secondary antibodies were incubated at room temperature for 60 min. Peroxidase activity was detected using the chemiluminescence method (Amersham) and visualized on X-Omat S films (Amersham). The assays were repeated three times.
3
Western Blot Analysis of Liver Proteins
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Liver tissues were homogenized with radioimmunoprecipitation assay lysis buffer containing phosphatase inhibitor cocktail and protease inhibitor. The extracted proteins were then separated by centrifugation at 12,000 rpm for 10 min at 4°C, resolved using 8%-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to the polyvinylidene difluoride membranes. After being blocked with 5% bovine serum albumin in Tris-buffered saline (TBS) at room temperature (RT) for 1 h, the blots were probed with a 1:1,000 or 1:2,000 dilution of the primary antibodies, including signal transducer and activator of transcription 3 (STAT3), phosphorylated STAT3 Tyr705 (Merck Millipore, MA, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Signalway Antibody Co., MD, USA) at 4°C overnight. Washed by TBS containing 1% Tween 20 (TBST), the membranes were then incubated with a 1:1,000 diluted enzyme-linked secondary antibodies at RT for 1 h. Washed by TBST again, the blots were then visualized using the chemiluminescence method (Amersham, PA, USA). Each protein band was quantified using Image J software (http://rsb.info.nih.gov), followed by densitometry reading, and undertaken either after normalization by GAPDH expression.
4
Protein Expression Analysis via Western Blot
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Proteins were extracted from the cells and subjected to SDS-PAGE. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). A polyclone rabbit anti-prohibitin antibody was purchased from Abcam (ab70672). Polyclonal rabbit anti-human PARP antibody, polyclonal rabbit anti-human caspase-3 antibody, polyclonal rabbit anti-human cleaved caspase-3 antibody, polyclonal rabbit anti-human caspase-9 antibody, polyclonal rabbit anti-human Bcl-2 antibody and polyclone rabbit anti-β-actin antibody were obtained from Cell Signaling Technology. The membranes were probed with all primary antibodies at a dilution of 1:1000, and secondary peroxidase-conjugated antibodies were used at a dilution of 1:2000. Bands were imaged using a chemiluminescence method (Amersham Biosciences, Sweden). Anti-β-actin antibody was used as an internal control.
5
Western Blot Analysis of Cellular Proteins
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Cells were washed once with ice-cold phosphate-buffered saline (PBS) and lysed with 0.1–0.2 ml RIPA lysis buffer. The protein concentration was determined by Coomassie protein assay (Pierce, Rockford, IL, USA). Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, Hercules, CA, USA), and transferred onto a nitrocellulose membrane (Amersham, Chalfont, UK). The membranes were incubated with primary antibodies (aPKCι, Santa Cruz Biotechnology, Santa Cruz, CA, USA; E-cadherin, Cell Signaling Technology, Danvers, MA, USA; Par6, Santa Cruz Biotechnology; β-catenin, Cell Signaling Technology; Vimentin, Cell Signaling Technology; N-cadherin, R&D Systems, Minneapolis, MN, USA; v-Ha-Ras, Santa Cruz Biotechnology; Caspase 3, Santa Cruz Biotechnology) at 4°C overnight. Peroxidase-conjugated secondary antibodies were applied for 60 min at room temperature at a dilution of 1 : 2000. β-Actin (Santa Cruz Biotechnology) was used as the internal control. Peroxidase activity was detected by using chemiluminescence method (Amersham) and visualized on X-Omat S films (Amersham). To quantify the relative levels of protein expression, the intensity of the specific bands was estimated by the ImageJ2X analysis software package (National Institute of Mental Health, Bethesda, MD, USA).
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