Tissue from the head of the pancreas obtained after control or NaT treatment as described previously was solubilized on ice in a lysis buffer consisting of NaCl (150 mmol/L), Tris, pH 7.5 (50 mmol/L), EDTA, pH 8.0 (10 mmol/L), dithiothreitol (1 mmol/L), Nonidet P-40 (1% v/v), sodium deoxycholate (0.5% w/v), sodium dodecyl sulfate (0.1% w/v), leupeptin (10 μg/mL), aprotinin (10 μg/mL), one tablet of protease inhibitors (cOmplete-Mini; Roche Diagnostics, Indianapolis, IN), and one tablet of phosphatase inhibitor (PhossSTOP; Roche Diagnostics) per 7 mL of solution. Supernates (10 μg protein) were loaded onto 4%–12% NuPage gels (Life Technologies), electrophoresed in 3-(N-morpholino)propanesulfonic acid buffer, and transferred onto polyvinylidene difluoride membranes (PerkinElmer, Waltham, MA). Blots were first reacted with a mouse monoclonal anti-5-LO antibody (BD Biosciences) at a dilution of 1/1000 and then with a rabbit polyclonal anti-α-tubulin antibody (GeneTex, Irvine, CA) at a dilution of 1/10,000. Bands were detected with the West Femto substrate (Thermo Scientific).
Anti α tubulin antibody
Manufactured by GeneTex
Sourced in United States
The Anti-α-tubulin antibody is a reagent used to detect α-tubulin, a key component of the cytoskeleton in eukaryotic cells. It can be used in various immunodetection techniques, such as Western blotting, immunocytochemistry, and immunohistochemistry, to visualize and study the distribution and organization of the microtubule network within cells.
Automatically generated - may contain errors
Lab products found in correlation
Complete mini, by Roche (2 mentions)
Nupage gel, by Thermo Fisher Scientific (2 mentions)
Phossstop, by Roche (2 mentions)
Anti 5 lo antibody, by BD (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by PerkinElmer (1 mentions)
Fv1200 confocal microscope, by Olympus (1 mentions)
Anti active β catenin antibody, by Merck Group (1 mentions)
Pvdf membrane, by PerkinElmer (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti α tubulin antibody
1
Quantifying 5-Lipoxygenase Protein Levels
2
Immunofluorescence Staining Protocol for α-Tubulin
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Immunofluorescence was conducted as described in our previous research (Peng et al., 2019 (link)). Specifically, the cells were fixed in 4% paraformaldehyde for 30 min and then treated with 0.3% Triton X-100 for 5 min. For non-specific blocking, the cells were incubated in 0.2% bovine serum albumin (Calbiochem, San Diego, CA, USA) for 15 min and then incubated with anti-α-tubulin antibody (dilution ratio was 1:100; GeneTex, Inc., North America, GTX628802) at 4°C for 12 h. The cells were stained with anti-mouse IgG fluorescent secondary antibody (dilution ratio was 1:200; Abways Biotechnology Co., Ltd., Shanghai, China, AB0132) for 2 h at room temperature. DNA was stained with Hoechst 33342 (Invitrogen). Fluorescence was imaged using OLYMPUS FV1200 confocal microscope (Olympus, Tokyo, Japan).
3
Western Blot Analysis of Wnt/β-Catenin Signaling
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A total of 1 million SW403 cells were seeded on 6-cm dishes overnight, and various compounds were treated for 48 h. The cell lysates were collected, and samples were loaded onto sodium dodecyl sulfate-polyacrylamide gel through electrophoresis and transferred onto polyvinylidene difluoride blotting membranes. The membranes were blocked using 5% skim milk with phosphate-buffered saline (PBST) for 1 h. An anti-Axin2 antibody (Cell Signaling Technology, Danvers, MA, USA) was added overnight in 4 °C conditions. The membrane was washed three times with PBST, and either an anti-active β-catenin antibody (Millipore, Burlington, MA, USA), anti-total β-catenin antibody (Genetex, Irvine, CA, USA), or anti-α-tubulin antibody (Genetex, Irvine, CA, USA) was added for 1 h. The membrane was then washed three times with PBST. An ECL reagent was added, and the luminescence was analyzed.
4
Protein Extraction and Western Blot Analysis
5
Immunofluorescence Staining Protocol
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After fixed in 4% paraformaldehyde for 30 min, cell samples were treated with 0.3% Triton X-100 for 3 min and blocked for 1 h with 0.2% bovine serum albumin (Calbiochem, San Diego, CA, United States). Anti-α-tubulin antibody (dilution ratio was 1:100; GeneTex, Inc. North America) was used as the primary antibodies. Anti-mouse IgG fluorescent secondary antibody (dilution ratio was 1:200) was purchased from Abways Biotechnology Co., Ltd. (Shanghai, China). DNA and mitochondria were stained with Hoechst 33342 (Invitrogen) and MitoTracker Green FM (Invitrogen), respectively.
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