Goat anti-mouse IgG conjugated to Alexa 594 and anti-rabbit IgG conjugated to Alexa 488 antibodies (Molecular Probes Inc., Eugene, OR, USA); mouse anti-α-tubulin monoclonal antibody and 4′,6-diamidino-2-phenylindole (DAPI) (Cell Signalling Technology, Danvers, MA, USA); rabbit anti-Caspase 3 polyclonal antibody, active (cleaved) form and Fluorsave (Merck Millipore, Darmstadt, Germany) were purchased from the indicated commercial sources. The protease inhibitor cocktail and all the remaining reagents were from Sigma-Aldrich (St. Louis, MO, USA).
Anti α tubulin mouse monoclonal antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-α-tubulin mouse monoclonal antibody is a laboratory reagent used for the detection and localization of α-tubulin, a key structural component of the cytoskeleton. This antibody can be used in various immunodetection techniques, such as Western blotting, immunofluorescence, and immunohistochemistry, to study the distribution and dynamics of the cytoskeleton in cells and tissues.
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Lab products found in correlation
Anti cyclin d1, by Cell Signaling Technology (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti foxo1, by Cell Signaling Technology (2 mentions)
Anti p27, by Cell Signaling Technology (2 mentions)
Anti p21, by Cell Signaling Technology (2 mentions)
Anti c myc, by Cell Signaling Technology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Cell Signaling Technology (1 mentions)
Goat anti mouse igg conjugated to alexa 594, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg conjugated to alexa 488, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Fluorsave, by Merck Group (1 mentions)
Ab174833, by Abcam (1 mentions)
Ab106814, by Abcam (1 mentions)
Ab106410, by Abcam (1 mentions)
Ab10983, by Abcam (1 mentions)
Mouse monoclonal anti β actin antibody a1978, by Merck Group (1 mentions)
Alexa 488 conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Confocal microscope, by Leica (1 mentions)
9 protocols using anti α tubulin mouse monoclonal antibody
1
Immunofluorescence Microscopy Assay
2
Protein Expression Analysis in Cell Lysates
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Cells were lysed in ice-cold lysis buffer, and the lysates were centrifuged at 12,000 × g at 4°C for 20 min. The sample concentration was evaluated by a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, MA, USA). Proteins were separated on SDS-polyacrylamide gels and then blotted onto membranes.[ 8 ] The blots were then reacted overnight at 4°C with the following primary antibodies: mouse anti-β-actin monoclonal antibody (A1978; Sigma-Aldrich), rabbit anti-CD137 monoclonal antibody (sc-398933; Santa Cruz Biotechnology, Inc.), mouse anti-HO-1 monoclonal antibody (ADI-OSA-110; Enzo Life Sciences, NY, USA), rabbit anti-FNDC5 monoclonal antibody (ab174833; Abcam, Cambridge, UK), goat anti-PGC1α polyclonal antibody (ab106814; Abcam), rabbit anti-PR domain containing 16 (PRDM16) polyclonal antibody (ab106410; Abcam), rabbit antiuncoupling protein 1 (UCP1) polyclonal antibody (ab10983; Abcam), and mouse anti-α-tubulin monoclonal antibody (#3873; Cell Signaling Technology, Danvers, MA, USA). Subsequently, the blot membranes were reacted with horseradish peroxidase-labeled secondary antibody for 1 h and further analyzed using enhanced chemiluminescence reagents (Pierce).
3
Paclitaxel-Induced Microtubule Dynamics
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Cells were plated on glass coverslips in a 12-well plate and was treated with 10 nM paclitaxel, or the same amount of DMSO, for 12 h after cell adherence. All groups were run in triplicate. The cells in each well were fixed with 4% paraformaldehyde (Sigma, USA) for 20 min, permeabilized in 0.3% Triton X-100 (Thermo Fisher, USA) for 10 min, blocked with 5% serum for 1-2 h, and then probed overnight at 4 ℃ with mouse monoclonal anti-α-tubulin antibody (Cell Signaling, 3873, 1:500). Next, the coverslips were washed, and the cells incubated with Alexa 488-conjugated goat anti-mouse secondary antibody (Thermo Fisher, A32723, 1:500) for 1 h at room temperature. Nuclei were then labeled with DAPI (Thermo Fisher, USA), and slides of different groups were observed with a confocal microscope (Leica, Germany).
4
Visualizing Microtubules and Mitotic Spindles
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MDA-MB231 cells were incubated with compounds for 6 h and 24 h to observe the cellular microtubule network and the mitotic spindles respectively. Thereafter, the cells were fixed and permeabilized as described in the past report [42 (link)]. After blocking nonspecific binding with 1 % donkey serum/PBS, the cells were incubated with the mouse monoclonal anti-α-tubulin antibody (Cell Signaling Technology) (1:1000 dilution) followed by the Alexa-488 conjugated anti-mouse IgG antibody (Invitrogen) (1:500 dilution). To visualized nuclei, the cells were incubated with Hoechst33342. For staining phospho-Histone H3, the fixed cells were treated with the rabbit monoclonal anti-phospho-Histone H3 (Ser10) antibody (Cell Signaling Technology) (1:1000 dilution) followed by the Alexa-594 conjugated anti rabbit IgG antibody (Invitrogen) (1:500 dilution). Cellular images were obtained with SP8 confocal microscopy (Leica).
5
Western Blotting of MYBL1 and TWIST1
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Western blotting was performed, according to a previously reported method.32 (link) The membranes were probed with polyclonal rabbit antibodies, anti-MYBL1 antibody (Abcam, Cambridge, MA, USA), and anti-TWIST1 (Cell Signaling Technology, Danvers, MA, USA). The membranes were then stripped and re-probed with an anti-α-tubulin mouse monoclonal antibody (Cell Signaling Technology) as a loading control.
6
Protein Expression Analysis by Western Blot
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Total protein was extracted from whole cells and 20 μg isolated protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was probed with anti-SOX9 (56KD) mouse monoclonal antibody (1:500; #ab76997, Abcam, Cambridge, MA, USA), anti-p-Akt(#4056), anti-Akt(#9272), anti-p21(#2947), anti-p27(#3686), anti-CyclinD1(#2922), anti-p-FOXO1(#9461), anti-FOXO1(#9454), anti-p-FOXO3(#9465) or anti-FOXO3(#2472) (Cell Signaling, Danvers, MA, USA). The membranes were then stripped and reprobed with an anti–α-tubulin mouse monoclonal antibody (1:1000; #2125; Cell Signaling, Danvers, MA, USA) as the loading control. Bound antibodies were visualized using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Dübendorf, Switzerland).
7
Western Blotting for APC, Cyclin D1, and c-MYC
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Western blotting was performed according to a previously reported method [17 (link)]. The membranes were probed with polyclonal mouse antibodies: anti-APC (ab15270; 1:1000; Abcam, Cambridge, UK), anti-cyclin D1 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and anti-c-MYC (1:1000; Cell Signaling Technology). The membranes were stripped and re-probed with anti-α-tubulin mouse monoclonal antibody (1:1000; Cell Signaling Technology) as the loading control.
8
Western Blot Analysis of Cell Signaling Proteins
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Total protein was extracted from whole cells by a previously described procedure. Lysate protein (20 μg) for Western blotting was separated by SDS-PAGE and electroblotted onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were probed with polyclonal rabbit antibodies against anti-FOXO1, anti-p21, anti-p27, anti-CyclinD1, and anti-Ki67 (1:1,000; Cell Signaling, Danvers, MA, USA). The membranes were stripped and re-probed with an anti-α-tubulin mouse monoclonal antibody (1:1,000; Cell Signaling) as a loading control.
9
Western Blotting for Protein Analysis
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Western blotting was performed according to a previously reported method [26] (link). The membranes were probed with polyclonal rabbit antibodies, anti-SOX7 (#ab80331, 1∶500; Abcam, Cambridge, MA, USA), anti-c-Myc (#9402), anti-CyclinD1 (#2978), anti-Rb (#9313) and anti-phosphorylated Rb (#9306; 1∶1,000; Cell Signaling, Danvers, MA, USA). The membranes were then stripped and re-probed with an anti-α-Tubulin mouse monoclonal antibody (#2125; 1∶1,000; Cell Signaling) as a loading control.
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