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Mouse monoclonal anti β tubulin

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The Mouse monoclonal anti-β-tubulin is a laboratory reagent used to detect and analyze the β-tubulin protein. It is a highly specific antibody that binds to the β-tubulin subunit of the tubulin cytoskeletal protein.

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Lab products found in correlation

Mops running buffer, by Thermo Fisher Scientific (3 mentions) Reblot plus strong antibody stripping solution, by Merck Group (3 mentions) Western blocking reagent, by Roche (3 mentions) Supersignal west pico detection reagent, by Thermo Fisher Scientific (3 mentions) Irdye conjugated secondary antibody, by LI COR (2 mentions) Iblot nitrocellulose transfer membrane, by Thermo Fisher Scientific (2 mentions) Protease and phosphatase 3 inhibitor cocktail, by Merck Group (2 mentions) Odyssey fluorescent scanning system, by LI COR (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Glial fibrillary acidic protein (gfap), by Merck Group (2 mentions) Lin28b, by Cell Signaling Technology (2 mentions) Lin28a a177, by Cell Signaling Technology (2 mentions) Msi1 n3c3, by GeneTex (2 mentions) Hnrnpa1, by Cell Signaling Technology (2 mentions) Mouse monoclonal anti β actin, by Merck Group (2 mentions) Goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions) Superscript 3 one step rt pcr system with platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions) Image pro plus software, by Media Cybernetics (1 mentions) Tcs sp8 confocal microscope, by Leica (1 mentions)

Spelling variants of this product

Anti-tubulin (542 citations) β-tubulin (395 citations) Anti-β-tubulin (260 citations) Mouse anti-tubulin (239 citations) Mouse anti-β-tubulin (117 citations) Mouse monoclonal anti-tubulin (34 citations) β-tubulin (mouse, (2 citations) Monoclonal mouse (2 citations) Mouse monoclonal anti-human β-tubulin (2 citations) Mouse monoclonal anti-β-tubulin antibody (T4026 (2 citations)

21 protocols using mouse monoclonal anti β tubulin

1

Imaging Microtubuli in PFA-Fixed HeLa Cells

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HeLa cell samples have been prepared using the PFA-fixation protocol outlined in [49 (link)]. Microtubuli have been stained using monoclonal mouse anti-β -tubulin (Sigma Aldrich) and goat anti-mouse IgG secondary antibody (ThermoFisher Scientific), labeled with Alexa Fluor 647 at 1:150 and 1:300 dilution, respectively. All imaging experiments have been conducted in imaging buffer prepared freshly from 150-200mM MEA (β-Mercapto-ethylamine hydrochloride) in PBS and pH adjusted to 7.4 using NaOH. The oxygen scavenging effect from MEA has been proven efficient enough to refrain from additional enzyme-based oxygen scavenger systems.
Diederich B., Then P., Jügler A., Förster R, & Heintzmann R. (2019). cellSTORM—Cost-effective super-resolution on a cellphone using dSTORM. PLoS ONE, 14(1), e0209827.
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2

Mcl-1 Isoform Expression Analysis

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HeLa cells were plated onto 35-mm plates. After transfection, total RNA was extracted using the phenol/chloroform method. All steps were performed at 4°C. RNA was quantified with a NanoDrop ND-1000, and protein/solvent contaminations were evaluated (A260/280 = 1.8 and A260/230 = 2.0). RNA was converted to cDNA and amplified using the Mcl-1 primers via RT-PCR (SuperScript III one-step RT-PCR system with Platinum Taq DNA polymerase; Invitrogen).
Proteins were separated by SDS–PAGE on a 4–12% precast gel and detected by Western blotting using polyclonal rabbit anti-Mcl-1 (1:500) for both the L (40 kDa) and S (32 kDa) isoforms (Santa Cruz Biotechnology) and monoclonal mouse anti–β-tubulin (1:5000; Sigma-Aldrich) as the primary antibodies and respective horseradish peroxidase (HRP)–labeled secondary antibodies (1:5000), according to standard protocols.
Morciano G., Giorgi C., Balestra D., Marchi S., Perrone D., Pinotti M, & Pinton P. (2016). Mcl-1 involvement in mitochondrial dynamics is associated with apoptotic cell death. Molecular Biology of the Cell, 27(1), 20-34.
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3

Quantifying CXCL12β in IL-1β-Treated BMECs

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Brain microvascular endothelial cells (BMECs) were treated with IL-1β (10 ng/ml) overnight and protein lysates were isolated using RIPA buffer supplemented with protease and phosphatase-3 inhibitor cocktail (Sigma). Lysates were resolved on a 4-12% Bis-Tris gel and transferred on to an iBlot Nitrocellulose transfer membrane (Invitrogen) according to standard protocols. Blots were probed with polyclonal rabbit anti-CXCL12β (eBioscinec) and monoclonal mouse anti-β-tubulin (Sigma) antibodies flowed by incubation with IRDye®-conjugated secondary antibodies (LI-COR). Blots were imaged using the Odyssey fluorescent scanning system (LI-COR) and analyzed using ImageJ.
Durrant D.M., Daniels B.P, & Klein R.S. (2014). IL-1R1 signaling regulates CXCL12-mediated T cell localization and fate within the CNS during West Nile virus encephalitis. Journal of immunology (Baltimore, Md. : 1950), 193(8), 4095-4106.
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4

Visualizing Cytoskeletal Structures in Cells

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Cells were seeded at 1 × 104 cells on an 8-well slide chamber under differentiation conditions for 5 to 7 days. After fixation with 4% PFA/PBS for 10 min and permeabilization with 0.2% Triton X-100 for 1 min, cells were incubated with monoclonal mouse anti β-tubulin (Sigma) at 4 °C overnight. The structure of intercellular β-tubulin and F-actin was visualized using a Leica TCS SP8 confocal microscope by triple-labeling with Alexa 563-coupled donkey anti-mouse secondary antibody (Thermo Fisher, Waltham, MA), Alexa 633-coupled phalloidin (Thermo Fisher), and DAPI respectively. The number of filopodia per cell and cells with nuclear lobulation were counted in at least 50 cells for each cell type. For nuclear area and protrusion analysis, images were processed using Image Pro plus software (Media Cybernetics, Rockville, MD). Fluorescent line scans of β-tubulin and F-actin were performed using the Leica TCS SP8 software along the vertical line in protrusions for YFP and YFP-WT cells and marginal fibrillary structures for YFP-Y64C and YFP-G12V. Line scans were conducted for at least 20 cells in each cell type.
Daimon E., Shibukawa Y., Thanasegaran S., Yamazaki N, & Okamoto N. (2021). Macrothrombocytopenia of Takenouchi-Kosaki syndrome is ameliorated by CDC42 specific- and lipidation inhibitors in MEG-01 cells. Scientific Reports, 11, 17990.
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5

CXCL12β Protein Expression Analysis

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The caudal CC was dissected and 10 µg protein lysates were isolated using RIPA buffer supplemented with a protease and phosphatase-3 inhibitor cocktail (Sigma-Aldrich). Lysates were resolved on a 4–12% Bis-Tris gel and transferred onto an iBlot Nitrocellulose transfer membrane (Invitrogen) according to standard protocols. Blots were probed with polyclonal rabbit anti-CXCL12β (eBioscience) and monoclonal mouse anti–β-tubulin (Sigma-Aldrich) antibodies, followed by incubation with IRDye-conjugated secondary antibodies (LI-COR). Blots were imaged using the Odyssey fluorescent scanning system (LI-COR) and analyzed using ImageJ (National Institutes of Health).
Williams J.L., Patel J.R., Daniels B.P, & Klein R.S. (2014). Targeting CXCR7/ACKR3 as a therapeutic strategy to promote remyelination in the adult central nervous system. The Journal of Experimental Medicine, 211(5), 791-799.
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6

Western Blotting of SMAD and RAS Proteins

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Western blotting was performed as previously described.29 (link) The primary antibodies used were anti-phospho-SMAD2 (Cell Signaling #3101), anti-SMAD2 (Invitrogen #51-1300; Carlsbad, CA), anti-SMAD2/3 (Cell Signaling #3102), anti-SMAD4 (Epitomics #1676-1; Burlingame, CA), anti-RAS (Santa Cruz Biotechnology), anti-β-actin (Sigma-Aldrich #A5441 Clone AC15), and anti-β-tubulin (mouse monoclonal; Sigma-Aldrich). Peroxidase-labeled anti-rabbit and anti-mouse secondary antibodies were purchased from Dako (mouse immunoglobulin G horseradish peroxidase–conjugated DAKO P0260, rabbit immunoglobulin G horseradish peroxidase–conjugated DAKO #P0448).
Chuvin N., Vincent D.F., Pommier R.M., Alcaraz L.B., Gout J., Caligaris C., Yacoub K., Cardot V., Roger E., Kaniewski B., Martel S., Cintas C., Goddard-Léon S., Colombe A., Valantin J., Gadot N., Servoz E., Morton J., Goddard I., Couvelard A., Rebours V., Guillermet J., Sansom O.J., Treilleux I., Valcourt U., Sentis S., Dubus P, & Bartholin L. (2017). Acinar-to-Ductal Metaplasia Induced by Transforming Growth Factor Beta Facilitates KRASG12D-driven Pancreatic Tumorigenesis. Cellular and Molecular Gastroenterology and Hepatology, 4(2), 263-282.
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7

Immunofluorescence Staining of Cytoskeletal Proteins

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Cells were fixed in 3% paraformaldehyde in PBS for 30 min., permeabilized with 0.2% Triton-X100 in PBS for 5 min., and blocked in 5% non-fat dry milk in PBS for 10 min; all done at room temperature. Washes in between were done with 10mM glycine in PBS. Incubation with primary antibodies was performed overnight at 4°C, followed by secondary antibody incubation for 30 min. at room temperature, both in blocking buffer. Nuclei were stained with Hoechst 33342 (Invitrogen, #H3570) 1:10 000 for 10 min. at room temperature. The following primary antibodies and dilutions were used: anti- vinculin mouse monoclonal antibody (Sigma, #V9131) 1:100, anti-β-tubulin mouse monoclonal (Sigma, #T4026) 1:200. The following secondary antibodies were used: goat anti-mouse Alexa Fluor 488 (Invitrogen, #A11017) 1:200, goat anti-mouse Alexa Fluor 546 (Invitrogen, #A11030) 1:500. Aqua Polymount (Polysciences) was used to mount coverslips on glass slides. Images were acquired using Olympus IX71 microscope, equipped with Olympus LUCPLanFLN objectives (20X NA 0.45, 40X NA 0.6, 60X NA 0.7) and a QuantiFire XI camera (Optronics).
Cichon M.A., Moruzzi M.E., Shqau T.A., Miller E., Mehner C., Ethier S.P., Copland J.A., Radisky E.S, & Radisky D.C. (2016). MYC is a crucial mediator of TGFβ-induced invasion in basal breast cancer. Cancer research, 76(12), 3520-3530.
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8

Western Blot Analysis of Neural Proteins

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Total protein samples (100μg per lane), isolated by sonication, were resolved by standard NuPAGE SDS-PAGE electrophoresis with MOPS running buffer (Life Technologies) and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with 1:10 Western Blocking Reagent (Roche) in TBS buffer with 0.1% of Tween-20 (TBST). The following day the membrane was incubated for 1h at RT with primary antibody solution in 1:20 Western Blocking Reagent diluted in TBST: rabbit polyclonal anti Lin28a (A177) (1:1000, Cell Signalling Technology), rabbit polyclonal anti Msi1 (N3C3) (1:1000, GeneTex), rabbit monoclonal anti hnRNP A1 (1:1000, D21H11) (Cell Signalling Technology), Lin28b (1:1000, Cell Signalling Technology), Tuj1 (1:20,000, GeneTex), GFAP (1:1000, SIGMA), DHX9 (1:1000, Protein-Tech), mouse-monoclonal anti–β-tubulin (1:10,000, Sigma). After washing in TBST, the blots were incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase and detected with SuperSignal West Pico detection reagent (Thermo Scientific). The membranes were stripped using ReBlot Plus Strong Antibody Stripping Solution (Chemicon) equilibrated in water, blocked in 1:10 western blocking solution in TBST and re-probed as described above. Full scans of the western blots presented in the manuscript are shown in the Supplementary Fig 10.
Nowak J.S., Choudhury N.R., de Lima Alves F., Rappsilber J, & Michlewski G. (2014). Lin28a regulates neuronal differentiation and controls miR-9 production. Nature communications, 5, 3687.
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9

LRRK2 and Tyrosine Hydroxylase Immunoblotting

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Following homogenization of brain samples in lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1X Complete protease inhibitor cocktail [Roche Applied Sciences], 1X phosphatase inhibitor cocktails 2 and 3 [Sigma]), samples were subjected to ultracentrifugation (100,000g for 30 min) and protein concentration of soluble supernatant fractions (NP-40-soluble) was determined by BCA protein assay (Pierce Biotechnology). Proteins (50 μg) we resolved on 7% SDS-PAGE gels and transferred to nitrocellulose membranes. Blots were probed with monoclonal antibodies against LRRK2 (MJFF4/c81-8, Epitomics; N241A/34, NeuromAbs, UC Davis), rabbit polyclonal anti-TH (Novus Biologicals), and mouse monoclonal anti-β-tubulin (Sigma Aldrich). Secondary antibodies used were mouse monoclonal anti-rabbit IgG, light chain-specific (Jackson Immunoresearch) and goat anti-mouse IgG, light chain-specific (Jackson Immunoresearch). All secondary antibodies were HRP-conjugated and were detected using ECL reagent (Amersham). Western blot images were captured on a chemiluminescent image analyzer (LAS-4000, Fujifilm) and protein bands were quantified by densitometric analysis using NIH ImageJ software.
Tsika E., Kannan M., Foo C.S., Dikeman D., Glauser L., Gellhaar S., Galter D., Knott G.W., Dawson T.M., Dawson V.L, & Moore D.J. (2014). Conditional Expression of Parkinson's Disease-Related R1441C LRRK2 in Midbrain Dopaminergic Neurons of Mice Causes Nuclear Abnormalities without Neurodegeneration. Neurobiology of disease, 71, 345-358.
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10

Western Blot Analysis of Neural Proteins

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Total protein samples (100μg per lane), isolated by sonication, were resolved by standard NuPAGE SDS-PAGE electrophoresis with MOPS running buffer (Life Technologies) and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with 1:10 Western Blocking Reagent (Roche) in TBS buffer with 0.1% of Tween-20 (TBST). The following day the membrane was incubated for 1h at RT with primary antibody solution in 1:20 Western Blocking Reagent diluted in TBST: rabbit polyclonal anti Lin28a (A177) (1:1000, Cell Signalling Technology), rabbit polyclonal anti Msi1 (N3C3) (1:1000, GeneTex), rabbit monoclonal anti hnRNP A1 (1:1000, D21H11) (Cell Signalling Technology), Lin28b (1:1000, Cell Signalling Technology), Tuj1 (1:20,000, GeneTex), GFAP (1:1000, SIGMA), DHX9 (1:1000, Protein-Tech), mouse-monoclonal anti–β-tubulin (1:10,000, Sigma). After washing in TBST, the blots were incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase and detected with SuperSignal West Pico detection reagent (Thermo Scientific). The membranes were stripped using ReBlot Plus Strong Antibody Stripping Solution (Chemicon) equilibrated in water, blocked in 1:10 western blocking solution in TBST and re-probed as described above. Full scans of the western blots presented in the manuscript are shown in the Supplementary Fig 10.
Nowak J.S., Choudhury N.R., de Lima Alves F., Rappsilber J, & Michlewski G. (2014). Lin28a regulates neuronal differentiation and controls miR-9 production. Nature communications, 5, 3687.
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