Anti α tubulin antibody dm1a

Manufactured by Merck Group

Sourced in Japan, Germany

Anti–α-tubulin antibody (DM1A) is a laboratory reagent used for the detection and analysis of α-tubulin, a key structural component of the cytoskeleton in eukaryotic cells. This antibody specifically binds to α-tubulin and can be used in various immunodetection techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to visualize and quantify the expression and localization of α-tubulin in biological samples.

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8 protocols using anti α tubulin antibody dm1a

Generating MCAK Phospho-Ser715 Antibody

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Anti-MCAK antibody was reported previously53 (link). To generate MCAK phospho-Ser715 antibody, a synthetic peptide containing phospho-Ser715 (CMQLEEQA-pS-RQISS) was conjugated to rabbit albumin (Sigma Chemical) and immunized into rabbits as described54 (link). The serum was collected by a standard protocol and preabsorbed by nonphosphorylated MCAK peptide (CMQLEEQASRQISS) followed by affinity-purification using CMQLEEQA-pS-RQISS-conjugated sulftone sepharose beads (Sigma Chemical). Other antibodies were obtained from commercial sources: mouse anti-PLK1 monoclonal antibody (Invitrogen); anti-phospho-Thr210 PLK1 antibody (BD Biosciences); anti-Cyclin B antibody (BD Biosciences); mouse anti-EB1 antibody (BD Biosciences); mouse monoclonal anti-GFP antibody (BD Biosciences); anti-α-tubulin antibody DM1A (Sigma-Aldrich).
MCAK siRNA targeting the 3′-UTR of MCAK gene was purchased from Qiagen (SI05040686). The small-hairpin RNA (shRNA) against MCAK was constructed using the same targeting sequences as its siRNA. To get a fluorescence-marked shRNA system, pLKO.1 cloning vector (Addgene) was reconstituted by inserting a sequence encoding mCherry-H2B to generate a plasmid co-expressing target shRNA and mCherry-H2B.

Shao H., Huang Y., Zhang L., Yuan K., Chu Y., Dou Z., Jin C., Garcia-Barrio M., Liu X, & Yao X. (2015). Spatiotemporal dynamics of Aurora B-PLK1-MCAK signaling axis orchestrates kinetochore bi-orientation and faithful chromosome segregation. Scientific Reports, 5, 12204.

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Analyzing APC/C Inhibition by Emi2 Fragment

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APC/C inhibition in cells expressing the ZBR–RL fragment of Emi2 was examined by comparing the CCRP levels between the AcGFP-Blank vector and the AcGFP-Emi2 fragment fusion transfectants. At 2 days post-transfection, the cells were collected, washed once with PBS, resuspended in lysis buffer [20 mM Tris–HCl buffer (pH 8.0), containing 1% Triton X-100, 150 mM NaCl, and protease inhibitor mixture (Roche Applied Science)], and incubated on ice for 30 min. The resulting cell lysates were centrifuged at 15,000 rpm for 15 min at 4 °C, fractionated by SDS–PAGE, and electroblotted onto a PVDF membrane. The membranes were subsequently blocked with skim milk prepared with TBS-Tween, and immunodetection was performed using the Immobilon Chemiluminescent Substrate Kit (Merck Millipore). To assess APC/C inhibition in cells expressing the AcGFP-Emi2 ZBR–RL, the following primary antibodies were employed for WB: anti-CycB1 antibody (V152; MBL) and anti-securin antibody (DCS-280; MBL) to measure endogenous CycB1 and securin levels, respectively; anti-GFP antibody (JL-8; Clontech, Takara) to monitor AcGFP fusion protein expression; and anti-α-tubulin antibody (DM1A; Sigma–Aldrich) as a loading control.

Shoji S., Muto Y., Ikeda M., He F., Tsuda K., Ohsawa N., Akasaka R., Terada T., Wakiyama M., Shirouzu M, & Yokoyama S. (2014). The zinc-binding region (ZBR) fragment of Emi2 can inhibit APC/C by targeting its association with the coactivator Cdc20 and UBE2C-mediated ubiquitylation. FEBS Open Bio, 4, 689-703.

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Investigating Lymphocyte Function-Associated Antigen-1 Activation

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Anti–mouse LFA-1 antibodies clones M17 and 2D7, mouse anti–human CD11a (Itgal), secondary antibodies, and ELISA kits were purchased from eBioscience. PE-conjugated anti–mouse CD11a clone M17, rat anti-mouse ART2.2 clone Nika102, and mouse anti–human activated LFA-1 clone 24 were purchased from BioLegend, Novus Biologicals, Abcam, and Hycult Biotech, respectively. CD45RB antibody was obtained from BD. Lipofectamine 2000 and mouse anti–human CD4 antibodies were purchased from Invitrogen. Cell-Tak, PMA, OVA, NAD, and anti–α-tubulin antibody (DM1A) were obtained from Sigma-Aldrich. Anti–Fascin-1 antibody (55K2) was purchased from Millipore and anti–α-actinin antibody was obtained from Cell Signaling Technology. For both mouse and human T reg assays, human recombinant IL-2 (injectable clinical preparation) was used.

Chen J., Ganguly A., Mucsi A.D., Meng J., Yan J., Detampel P., Munro F., Zhang Z., Wu M., Hari A., Stenner M.D., Zheng W., Kubes P., Xia T., Amrein M.W., Qi H, & Shi Y. (2017). Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy. The Journal of Experimental Medicine, 214(2), 327-338.

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Immunofluorescence Staining Antibodies

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For primary antibodies, we used anti-KIFC3 antibody (H-300; Santa Cruz Biotechnology), anti-USP47 antibody (4E7; Santa Cruz Biotechnology; ab72143; Abcam, Cambridge, UK), anti-GFP antibody (598, Medical and Biological Laboratories, Nagoya, Japan; GF090R, Nacalai Tesque), anti–E-cadherin antibodies (H-108; Santa Cruz Biotechnology; SHE78-7 and HECD-1; Takara Bio, Shiga, Japan; 36; BD Biosciences, San Jose, CA; 24E10; Cell Signaling Technology, Danvers, MA), anti-CBLL1/HAKAI antibody (Proteintech Group, Chicago, IL), anti-PLEKHA7 antibody (Sigma-Aldrich), anti–p120-catenin antibody (BD Biosciences), anti–α-tubulin antibody (DM1A, Sigma-Aldrich; Abcam), anti-LAMP1 antibody (Santa Cruz Biotechnology), and anti-FLAG antibody (M2, Sigma-Aldrich). Rabbit polyclonal anti-CAMSAP3 antibody was produced previously (Tanaka et al., 2012 (link)). For secondary antibodies, we used Alexa 488–conjugated anti-mouse secondary antibody, Alexa 555–conjugated anti-mouse secondary antibody, Alexa 568–conjugated anti-mouse secondary antibody, Alexa 488–conjugated anti-rabbit secondary antibody, Alexa 568–conjugated anti-rabbit secondary antibody, Alexa 647–conjugated anti-rabbit secondary antibody, and Alexa 488–conjugated anti-rat secondary antibody (Life Technologies).

Sako-Kubota K., Tanaka N., Nagae S., Meng W, & Takeichi M. (2014). Minus end–directed motor KIFC3 suppresses E-cadherin degradation by recruiting USP47 to adherens junctions. Molecular Biology of the Cell, 25(24), 3851-3860.

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Microtubule Binding Protein Characterization

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MBP or MBP-Mora were spun at 100,000 g for 15 min in an Optima MAX ultracentrifuge (Beckman) to remove any insoluble material. Then, 10 µl of the sample was incubated for 15 min at 37°C in general tubulin buffer (GTB; Cytoskeleton Inc.) with 2.25 mg/ml 99% pure bovine tubulin (Cytoskeleton Inc.) and 1 mM GTP. The sample was incubated for a further 10 min at 37°C in the presence of 100 µM taxol (+). A negative control (−) was run in parallel, with an incubation temperature of 4°C and taxol replaced with GTB. The samples were layered onto a cushion of GTB with 40% glycerol and centrifuged at 100,000 g for 45 min at 4°C. The pellet and supernatant fractions were collected individually, and proteins present in each fraction were determined by western blot analysis. The supernatant was collected and an equal volume of buffer used to resuspend the pelleted microtubules and interacting protein. Equivalent volumes of supernatant and pellet were run side-by-side using SDS-PAGE, and analysed by western blotting using anti-MBP (NEB) and anti-α-Tubulin antibody (DM1A; Sigma-Aldrich).

Palumbo V., Tariq A., Borgal L., Metz J., Brancaccio M., Gatti M., Wakefield J.G, & Bonaccorsi S. (2020). Drosophila Morgana is an Hsp90-interacting protein with a direct role in microtubule polymerisation. Journal of Cell Science, 133(2), jcs236786.

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Western Blot Analysis of Chicken RCC1 Protein

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Whole-cell lysates were prepared and analyzed by 5–20% SDS–PAGE. Western blot analysis was performed by a standard protocol. Rabbit polyclonal anti-chicken RCC1 antibody (used at 1:10,000) was raised against hexahistidine-ggRCC1 and affinity purified. Anti-Ran antibody (used at 1:5000; BD Transduction Laboratories, Tokyo, Japan) and anti–α-tubulin antibody (DM1A, used at 1:1000; Sigma-Aldrich) were used. Anti–histone H3 antibody (used at 1:10.000) was a gift from H. Kimura (Tokyo Institute of Technology, Tokyo, Japan).

Furuta M., Hori T, & Fukagawa T. (2016). Chromatin binding of RCC1 during mitosis is important for its nuclear localization in interphase. Molecular Biology of the Cell, 27(2), 371-381.

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Histone Deacetylase Enzyme Assay

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Dimethyl sulphoxide (DMSO), percoll, Nonidet P-40 (NP40), fetal bovine serum (FBS), thimerosal, gambogic acid, trichostatin A (TSA), bovine serum albumin (BSA), carmine-red and Canada balsam were purchased from Sigma-Aldrich (Saint Lous, USA); CellTiter-Glo (CTG) reagent from Promega (Madison, USA); Dulbecco-Modified Eagle’s Medium (DMEM) with or without phenol red, HEPES, L-glutamine from Lonza (Basel, Switzerland); antibiotic-antimycotic reagent (100×) from Thermo Fisher Scientific (Waltham, USA); HDAC1 enzyme (BMLSE456), substrate (BML-KI104) and developer solution BML-KI105) from Enzo Life Sciences, Inc (Farmingdale, USA); Dacinostat (S1095) from Selleckchem (Munich, Germany); the primary monoclonal anti-α-tubulin antibody (DM1A) from Sigma-Aldrich; the anti-acetylated-lysine (Ac-K2-100) from Cell Signaling Technology (Danvers, USA); goat anti-mouse and anti-rabbit IgG (H+L)-horseradish peroxidase secondary antibodies from Bio-Rad Laboratories (Hercules, USA).

Guidi A., Saccoccia F., Gennari N., Gimmelli R., Nizi E., Lalli C., Paonessa G., Papoff G., Bresciani A, & Ruberti G. (2018). Identification of novel multi-stage histone deacetylase (HDAC) inhibitors that impair Schistosoma mansoni viability and egg production. Parasites & Vectors, 11, 668.

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Immunostaining and Immunoblotting with Tubulin Antibodies

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Anti α-tubulin antibody (DM1A) and anti α-tubulin antibody (B-5-1-2) were purchased from Sigma-Aldrich and used at ×1,000 dilution for immunostaining (DM1A and B-5-1-2) and immunblotting (B-5-1-2). Anti β-tubulin antibody (AA2) was purchased from Abcam and used at ×1,000 dilution for immunostaining and immunoblotting. Anti polyE antibody was purchased from AdipoGen Life Sciences (AG25B-0030-C050) and used at ×5,000 dilution for immunoblotting and ×10,000 dilution for immunostaining. Alexa Fluor 488 plus goat anti-mouse IgG (A32723) and Alexa Fluor 594 plus goat anti-rabbit IgG (A32740) were purchased from Thermo Fisher Scientific and used at ×1,000 dilution for immunostaining. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Minamino N., Norizuki T., Mano S., Ebine K, & Ueda T. (2022). Remodeling of organelles and microtubules during spermiogenesis in the liverwort Marchantia polymorpha. Development (Cambridge, England), 149(15).

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