Transfected COS cells were scraped and lysed in lysis buffer (100 mM NaCl, 40 mM KCl, 1 mM EDTA, 20 mM HEPES, 10% Glycerol, 1% Triton-X, with Complete Protease Inhibitor from Roche) for 1 h at 4 °C. Lysates were cleared by 10 min centrifugation at 20,000 g. For deglycosylation, solubilized proteins were incubated with 250 U EndoH and/or 10 U PngaseF (Promega, France) at 37 °C overnight. Before separating proteins on a 10% polyacrylamide gel and blotting onto PVDF membrane (Hybond-P, Amersham Bioscience), lysates were boiled 5 min at 99 °C with Laemmli buffer containing reducing ß-Mercaptoethanol. Blots were blocked with 5% milk/PBS for 1 h and incubated with anti-TWIK2 polyclonal antibody (1:200, Alomone, Jerusalem, Israel) at 4 °C overnight or with anti-β-Tubulin monoclonal antibody (1:1000, clone TUB 2.1, Sigma-Aldrich, France) 1 h at RT. Secondary horseradish peroxidase coupled antibodies were diluted 1:10.000 and incubated 1 h at RT before detection by enhanced chemiluminescence reaction (ECL, Amersham Biosciences).
Anti β tubulin monoclonal antibody
Manufactured by Merck Group
Sourced in Germany
The Anti-β-tubulin monoclonal antibody is a laboratory reagent used in various research applications. It specifically recognizes and binds to the β-tubulin protein, which is a critical component of the cytoskeleton in eukaryotic cells. This antibody can be utilized in techniques such as immunoblotting, immunohistochemistry, and immunocytochemistry to detect and study the expression and localization of β-tubulin within biological samples.
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Lab products found in correlation
Hoechst 33342, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Euroclone (2 mentions)
Mouse monoclonal anti tdo antibody, by Novus Biologicals (2 mentions)
High d glucose dmem, by Euroclone (2 mentions)
Tri reagent, by Merck Group (2 mentions)
Endo h, by Promega (1 mentions)
Clone tub 2, by Merck Group (1 mentions)
Hybond p, by Cytiva (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Pngase f, by Promega (1 mentions)
Anti cx43 polyclonal antibody, by Merck Group (1 mentions)
Bx 51w1i upright microscope, by Olympus (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti ahr, by Cell Signaling Technology (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Propidium iodide, by Santa Cruz Biotechnology (1 mentions)
Anti rhoa, by Cell Signaling Technology (1 mentions)
17 protocols using anti β tubulin monoclonal antibody
1
Deglycosylation and Western Blot Analysis
2
Immunostaining of Cytoskeletal and Junctional Proteins
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Cells grown on glass coverslips were fixed at room temperature with 2% paraformaldehyde for 30 min and then washed three times with PBS. Then, cells were incubated three times for 5 min in 0.1 M PBS-glycine, followed by 30 min incubation with 0.1% PBS-Triton X-100 containing 10% NGS. The permeabilized cells were incubated with anti-β-tubulin monoclonal antibody (Sigma, 1:400) and anti-Cx43 polyclonal antibody (SIGMA, 1:400) diluted in 0.1% PBS-Triton X-100 with 2% NGS at 4°C overnight. After five rinses in 0.1% PBS-Triton X-100, cells were incubated with goat anti-mouse IgG Alexa Fluor 555 (1:1,000), goat anti-rabbit IgG Alexa Fluor 488 (1:1,000), or Alexa Fluor 488-phalloidin at room temperature for 50 min. After several rinses, coverslips were mounted in DAPI Fluoromount-G medium and examined with an Olympus BX 51W1I upright microscope with a 40× water immersion objective or a confocal laser-scanning microscope with a 63× objective (Olympus, Fluoview FV1000, Tokyo, Japan).
3
Protein Immunoblotting of SK-Mel-28 Cells
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SK-Mel-28 cells were lysed in a Triton® X-100 lysis buffer, pH 7.4, followed by a centrifugation at 14,000× g rpm for 10 min at 4 °C [57 (link)]. Cell lysate was run on 10% SDS-polyacrylamide gel electrophoresis, blotted onto PVDF membrane (Merck-Millipore, Darmstadt, Germany), and immunostained with anti-phospho Akt (1:1000; Cell Signaling Technology, MA, USA), anti-AHR (1:1000; Cell Signaling Technology), and anti β-tubulin monoclonal antibody (1:1000; Sigma-Aldrich, MO, USA). The antigen–antibody complexes were visualized using appropriate secondary antibodies and the ECL detection system by means of ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA)
4
Molecular Regulation of Cellular Dynamics
5
Immunofluorescence Staining and Imaging Protocol
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Antibody and phalloidin stainings were performed as described previously [25 (link)]. The samples were imaged with a 63x magnification (oil immersion) using a Leica TCS-SP2 confocal microscope and the LCS software. The primary antibodies used in this study were the following: rabbit polyclonal against D. melanogaster Ref(2)P protein [54 (link)], mouse monoclonal against Flag tag (Clone M2, Sigma-Aldrich) and rabbit monoclonal anti-Cathepsin L (ab133641, Abcam). The appropriate Cy3-conjugated secondary antibodies were purchased from Jackson Immunoresearch Laboratories.
Lysotracker staining on tissue was performed as in ref. [55 (link)]. Images were obtained with a fluorescence microscope (Nikon Eclipse 90i) controlled by Nikon Software (Universal Imaging Corp.) using a 60x Plan-Neofluor oil objective.
Image analysis and processing were done with Fiji/ImageJ (National Institute of Health) and Photoshop CS6 (Adobe).For experiments carried out in HeLa cells, the following antibodies were used: anti-β-tubulin monoclonal antibody (Sigma-Aldrich), anti-p62 monoclonal antibody (H00008878-M01, Novus Biologicals), anti-UBPY. Dimethyl sulfoxyde (DMSO) and Hoechst #33342 were from Sigma-Aldrich and Bafilomycin A1 (#tlrl-baf1) was purchased from Invivogen.
Lysotracker staining on tissue was performed as in ref. [55 (link)]. Images were obtained with a fluorescence microscope (Nikon Eclipse 90i) controlled by Nikon Software (Universal Imaging Corp.) using a 60x Plan-Neofluor oil objective.
Image analysis and processing were done with Fiji/ImageJ (National Institute of Health) and Photoshop CS6 (Adobe).For experiments carried out in HeLa cells, the following antibodies were used: anti-β-tubulin monoclonal antibody (Sigma-Aldrich), anti-p62 monoclonal antibody (H00008878-M01, Novus Biologicals), anti-UBPY. Dimethyl sulfoxyde (DMSO) and Hoechst #33342 were from Sigma-Aldrich and Bafilomycin A1 (#tlrl-baf1) was purchased from Invivogen.
6
Visualizing Spindle and Chromosomes in Blastomeres
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After blastomeres had fused with PB2s and reached the first metaphase, they were processed for visualization of the spindle and chromosomes.
Blastomeres were placed in acidic Tyrode's solution (pH 2.5; Sigma-Aldrich) for 20-30 min, and permeabilized with 1% triton X-100 (Sigma-Aldrich) in Dulbecco's phosphate-buffered saline (D-PBS, pH 7.4) for 1 h at room temperature. After washing with D-PBS containing 0.3 % bovine serum albumin (BSA; Fraction-V, Sigma-Aldrich) (D-PBS/BSA), blastomeres were incubated with anti-β-tubulin monoclonal antibody (1:100 dilution; Sigma-Aldrich) in D-PBS for 1-2 h at 37°C. The cells were stored at 4°C in D-PBS/BSA overnight. The cells were then incubated in D-PBS containing an anti-mouse IgG-FITC-conjugated antibody (1:400 dilution; Sigma-Aldrich)
for 1 h at 37°C in dark. Finally, the cells were mounted on a glass slide and covered with a mounting medium containing 4′-6-diamidino-2-phenylindole (DAPI) (Vectashield; Vector Laboratories, Burlingame, CA, USA) for fluorescent microscopy.
Blastomeres were placed in acidic Tyrode's solution (pH 2.5; Sigma-Aldrich) for 20-30 min, and permeabilized with 1% triton X-100 (Sigma-Aldrich) in Dulbecco's phosphate-buffered saline (D-PBS, pH 7.4) for 1 h at room temperature. After washing with D-PBS containing 0.3 % bovine serum albumin (BSA; Fraction-V, Sigma-Aldrich) (D-PBS/BSA), blastomeres were incubated with anti-β-tubulin monoclonal antibody (1:100 dilution; Sigma-Aldrich) in D-PBS for 1-2 h at 37°C. The cells were stored at 4°C in D-PBS/BSA overnight. The cells were then incubated in D-PBS containing an anti-mouse IgG-FITC-conjugated antibody (1:400 dilution; Sigma-Aldrich)
for 1 h at 37°C in dark. Finally, the cells were mounted on a glass slide and covered with a mounting medium containing 4′-6-diamidino-2-phenylindole (DAPI) (Vectashield; Vector Laboratories, Burlingame, CA, USA) for fluorescent microscopy.
7
Fluorescent Indole Cytotoxicity Assay
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680C91 ((E)-6-fluoro-3-[2-(3-pyridyl)vinyl]-1H-indole), ribonuclease-A, CHPx, anti β-tubulin monoclonal antibody, TRI Reagent were from Merck (KGaA, Darmstadt, Germany); Annexin V was from ImmunoTools GmbH (Gladiolenweg 2; Germany); mouse monoclonal anti-TDO antibody was from NovusBio (Bio-Techne SRL, MI, Italy); propidium iodide (PI) (BD Biosciences); high D-glucose DMEM, RPMI 1640, M199 and PBS were from Euroclone S.p.A. (MI, Italy).
8
Inhibition of IDO1 and TDO in Cell Studies
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The 680C91 ((E)-6-fluoro-3-[2-(3-pyridyl)vinyl]-1H-indole), 1-MT, a competitive inhibitor of IDO1, anti β-tubulin monoclonal antibody, TRI Reagent, and actinomycin-D were from Merck (KGaA, Darmstadt, Germany). Mouse monoclonal anti-TDO antibody was from NovusBio (Bio-Techne, Minneapolis, MI, USA). high D-glucose DMEM, and PBS were from Euroclone S.p.A. (Pero, Milan, Italy). Epacadostat was from DivBioScience, Ulvenhout, The Netherlands.
9
Monopolar Spindle Induction Assay
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HeLa cells (5 × 103 cells/well) were seeded in 96-well plates (Corning, 3603). Drugs were diluted appropriately in medium from 10 mM stocks in 100% DMSO and added to the cells. Following 8 hours incubation with drugs, cells were fixed with 4% paraformaldehyde (20 minutes at room temperature), permeabilized with PBST (0.5% Triton X-100 in PBS) for 15 minutes, and washed before blocking. Cells were blocked with 4% BSA for at least 30 minutes at room temperature and stained with anti-β-tubulin monoclonal antibodies (Sigma) at 400-fold dilution for 1 hour with Alexafluor 488-conjugated goat anti-mouse secondary antibodies (Invitrogen) at 300-fold dilution for 1 hour. DNA was detected with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). Actin was detected with Phalloidin. High content screening (Perkin Elmer Operetta) was used to photograph stained cells at 40× magnification, and Columbus software was used for statistical analysis of the percentage of mitotic cells with monopolar spindles present in treated cells over the total number of cells in mitosis counted after 8 hours incubation with drugs.
10
Oxidase Knockout Mice Protocol
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Fetal calf serum, penicillin/streptomycin, and Trizol reagent were from GIBCO (Grand Island, NY, USA). Poly-L-lysine (mol. wt. > 300,000), trypsin, DNAse, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), propidium iodide (PI), cytochrome c, staurosporine, and 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) were from Sigma (St. Louis, MO, USA). Dihydroethidium and calcein-AM were purchased from Molecular Probes, Invitrogen (Carlsbad, CA, USA). Mouse anti-actin monoclonal antibodies were from Chemicon Int., anti-β-tubulin monoclonal antibodies were from Sigma-Aldrich, and FITC-goat anti-mouse IgG conjugated were from Zymed Laboratories Inc. All other chemicals were of the purest grade available from regular commercial sources. The colonies of NOX2 (gp91phox) knockout mice and NOX3 knockout mice on a C57BL6 background were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and were bred in the animal house of our institute.
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