Cells in each group were washed with ice-cold phosphate-buffered saline and lysed in RIPA solution containing protease inhibitor cocktail. Protein concentration was determined with BCA method. A total of 100 μg of protein was added in 12% sodium dodecyl sulfate-polyacrylamide gel, and then transferred to polyvinylidene difluoride membrane. The membrane was blocked with 5% non-fat milk for 4 h, then incubated with primary antibody overnight at 4 °C (rabbit polyclonal antibody against RARα; 1:1000; Santa Cruz, SC-551, USA) and then with secondary antibody (goat anti-rabbit antibody, 1:1000, Zhongshan Goldenbridge Biotechnology Co. Ltd., Beijing, China [ conjugate]) for 1 h at 37 °C. After washing with Tris-Buffered Saline Tween-20 (TBST), the autoradiograms were scanned and subjected to densitometry. β-actin (mouse monoclonal antibody against β-actin, 1:500; Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China) was used as an internal control. Protein bands were visualized using the Quantity One Software (BIO-RAD, USA).
Goat anti rabbit antibody
Manufactured by Zhongshan Golden Bridge Biotechnology
Sourced in China, United States
The Goat anti-rabbit antibody is a secondary antibody that binds to rabbit primary antibodies. It is commonly used in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry, to detect and quantify target proteins or molecules in biological samples.
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Lab products found in correlation
5 protocols using goat anti rabbit antibody
1
Western Blot Analysis of RARα Protein
2
Immunofluorescence Analysis of Autophagy and NF-κB
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Cells were washed three times with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 15 minutes, and blocked with bovine serum albumin (BSA) at 37°C for 30 minutes. After washing three times with PBS, the cells were incubated with anti-LC3B (1:100, Cell Signaling Technology, Danvers, MA, USA), p-IKBα (1:200, GeneTex, USA) and p-NF-κBp65 (1:1000, Abcam), overnight at 4°C, followed by goat anti-rabbit antibody (1:200 Zhongshan Golden Bridge, Beijing, China) for 1 hour at 37°C in the dark. The cells were counterstained with DAPI (Yusen Biotech Inc, Shanghai, China) for 1 hour. Finally, the positive cells was analyzed by confocal fluorescence microscopy (Olympus Tokyo, Japan).
3
Westenblotting for Akt1, β-catenin
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1x106 cells in each group were washed with ice-cold phosphate-buffered saline and lysed in RIPA solution containing protease inhibitor cocktail. Protein concentration was determined with BCA method. A total of 80 μg of protein was added in 8% sodium dodecyl sulfate-polyacrylamide gel, and then transferred to polyvinylidene difluoride membrane. The membrane was blocked with 5% non-fat milk for 2 h, then incubated with specific antibodies(monoclonal antibody against Akt1;1:1000; Abcam,Hong Kong,China, monoclnal antibodies against β-catenin, GSK3β, p-GSK3-β (S9), c-Myc; 1:1000;Cell Signaling Technology,USA) overnight at 4 °C and then with secondary antibody(goat anti-rabbit antibody, 1:1000, Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China) for 1 h at 37 °C. After washing with Tris-Buffered Saline Tween-20 (TBST), the autoradiograms were scanned and subjected to densitometry. β-actin(mouse monoclonal antibody against β-actin, 1:500; Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China) was used as an internal control. Protein bands were visualized using the Quantity One Software (BIO-RAD, USA). Each experiment was repeated at least three times.
4
Nonylphenol Gavage in Rats
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Nonylphenol for gavage (purity of 99%) was purchased from the Tokyo Chemical Co, Ltd (Tokyo, Japan). Rat ChAT and AchE kits were purchased from the Nanjing Jiancheng biological technology Co, LTD (NanJing, China). Anti-mouse-UL trapture antibody produced in 5 MG rabbit were obtained from the Beijing Zhongshan Biotechnology Reagents Co, Ltd. (Beijing, China). Goat anti-rabbit antibody was purchased from the Beijing Zhongshan Golden Bridge Biotechnology Co, Ltd. (Beijing, China). diaminobenzedine (DAB) chromogenic kit was purchased from the Dako Co (Glostrup, Denmark). All other chemicals were commercially available.
5
Western Blot Analysis of Signaling Proteins
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Cells in each group were washed with ice-cold phosphate-buffered saline (PBS) three times and lysed in RIPA solution containing protease inhibitor phenylmethanesulfonyl fluoride (PMSF), phosphatase inhibitor NaF and Na3VO3. Protein concentration was measured by BCA method. 50 μg total protein was added in 10% sodium dodecyl sulfate-polyacrylamide gel and then transfered to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk for 1 hour and incubated with specific antibodies(polyclonal antibody against p-p38α MAPK; 1:1000; Millipore, USA; polyclonal antidoby against p38α MAPK, HA-Tag; 1:1000; CST, USA; monoclonal antibody against Myc-Tag; 1:1000; CST, USA; polyclonal antibody against RARα; 1:1000; Santa Cruz, USA; polyclonal antibody against C/EBPβ, CD11b; 1:500; Wanleibio; China) overnight at 4 °C and then with secondary antibody(goat anti-rabbit antibody, 1:5000 and goat anti-mouse antibody, 1:2000; Zhongshan Goldenbridge Biotechnology Co. Ltd., Beijing, China) for 1 h at 37 °C. After washing with Tris-Buffered Saline Tween-20 and Tris-Buffered Saline (TBST and TBS), the autoradiograms were scanned and subjected to densitometry. β-actin (monoclonal antibody against β-actin, 1:1000; Zhongshan Goldenbridge Biotechnology Co. Ltd.,Beijing, China) was used as an internal control.
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