Mouse anti α tubulin dm1α

Manufactured by Merck Group

Sourced in United Kingdom

Mouse anti-α-tubulin DM1α is a monoclonal antibody that specifically recognizes the α-tubulin subunit of microtubules. It can be used for the detection and localization of α-tubulin in various research applications.

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α-tubulin (1242 citations) Anti-α-tubulin (920 citations) Anti-tubulin (542 citations) Mouse anti-α-tubulin (406 citations) Mouse anti-tubulin (239 citations) Mouse α-tubulin (32 citations) Anti-tubulin DM1α (8 citations) Anti-α-tubulin (DM1α, (5 citations) Mouse anti-tubulin DM1α (4 citations) α-tubulin (DM1α) (3 citations)

14 protocols using mouse anti α tubulin dm1α

Western Blot Protein Detection

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Samples were run on an SDS-PAGE gel, and then proteins were transferred to low-fluorescence PVDF membrane (catalogue number GE10600022, GE Health and Life Sciences). Membranes were blocked in 5% milk in 1X Tris-buffered saline (TBS) with 0.1% Tween-20 (0.1% TBST) for 1 hour at room temperature and incubated with primary antibody in block overnight at 4°C. After washing in 0.1% TBST membranes were incubated with secondary antibodies for 2–4 hours at room temperature. Membranes were then imaged using either chemiluminescence (Super Signal West Femto ECL, catalogue number 34094, ThermoFisher Scientific) or fluorescence. The following antibodies were used for western blotting: mouse anti-α-tubulin DM1α (1:1000, Sigma-Aldrich), mouse anti-actin C4 (1:1000, Sigma-Aldrich), rabbit anti-Ac-K394 (1:50, this study), Cy5 anti-mouse (1:10000, Jackson ImmunoResearch, West Grove, PA), and HRP-conjugated anti-rabbit (1:10000, Bio-Rad Laboratories).

Saunders H.A., Johnson-Schlitz D.M., Jenkins B.V., Volkert P.J., Yang S.Z, & Wildonger J. (2022). Acetylated α-tubulin K394 regulates microtubule stability to shape the growth of axon terminals. Current biology : CB, 32(3), 614-630.e5.

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Immunofluorescence Analysis of PRC1 in PtK2 Cells

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To quantify the region of PRC1 enrichment in the metaphase spindle in PtK2 cells (Figure 4A–B) and confirm PRC1 depletion following RNAi in PtK2 cells (Figure 4C, Figure 4—figure supplement 1B), cells were fixed with 95% methanol + 5 mM EGTA at −20°C for 1 min, washed with TBS-T (0.1% Triton-X-100 in TBS), and blocked with 2% BSA in TBS-T for 1 hr. Primary and secondary antibodies were diluted in TBS-T+2% BSA and incubated with cells for 1 hr (primary) and for 25 min at room temperature (secondary). DNA was labeled with Hoescht 33342 (Sigma, St. Louis, MO) before cells were mounted in ProLongGold Antifade (P36934; Thermo Fisher). Cells were imaged using the spinning disk confocal microscope described above. Antibodies: rabbit anti-PRC1 (1:100, BioLegend, San Diego, CA), mouse anti-α-tubulin DM1α (1:1000, Sigma-Aldrich), anti-mouse secondary antibodies (1:500) conjugated to Alexa Fluor 488 (A11001; Invitrogen), anti-rabbit secondary antibodies (1:500) conjugated to Alexa Fluor 647 (A21244; Life Technologies).
We observed PRC1 intensity at spindle poles but this intensity is similar in both mock RNAi (Luciferase) and PRC1 RNAi cells (0.80 ± 0.07 in mock RNAi vs. 0.86 ± 0.08 in PRC1 RNAi (AU), n = 20 cells, p=0.61, Mann-Whitney U test), suggesting non-specific antibody labeling by the human PRC1 antibody at spindle poles in PtK2 rat kangaroo cells.

Suresh P., Long A.F, & Dumont S. (2020). Microneedle manipulation of the mammalian spindle reveals specialized, short-lived reinforcement near chromosomes. eLife, 9, e53807.

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Immunofluorescence and Western Blotting Antibodies

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The following primary antibodies were used for immunofluorescence: rabbit anti-Asl (Conduit and Raff, 2010 (link)), guinea-pig anti-Asl (Roque et al., 2012 (link)), rat anti-Asl (Franz et al., 2013 (link)), mouse anti-GTU88* (Sigma-Aldrich), mouse anti-α-tubulin (DM1 α; Sigma-Aldrich), rabbit anti-Cnn (Lucas and Raff, 2007 (link)), sheep anti-Cnn (Conduit et al., 2014a (link)), rabbit anti-Ana1CT (Stevens et al., 2010a (link)), rabbit anti-Ana1Mid (Conduit and Raff, 2010 (link)), guinea-pig anti-Ana1 (Conduit et al., 2014b (link)), rat anti-Ana1CT (this study), rabbit anti-Sas4 (Basto et al., 2006 (link)) and rabbit anti-Spd-2 (Dix and Raff, 2007 (link)) antibodies, all used at 1:500 (see details in Table S1). Secondary antibodies conjugated to Alexa Fluor 405, 488, 568, 594 (used for SIM) and 647 (Invitrogen) were used, all at 1:1000; GFP-booster–atto488 (ChromoTek) was used at 1:500. DNA was labelled with Hoechst 33342 (Invitrogen). Rabbit anti-Ana1 (Conduit et al., 2010 (link)) and mouse anti-actin (Sigma-Aldrich) antibodies, and appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare) were used for western blotting (all 1:3000).

Saurya S., Roque H., Novak Z.A., Wainman A., Aydogan M.G., Volanakis A., Sieber B., Pinto D.M, & Raff J.W. (2016). Drosophila Ana1 is required for centrosome assembly and centriole elongation. Journal of Cell Science, 129(13), 2514-2525.

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Comprehensive Immunofluorescence Staining Protocol

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Cells were fixed in either 4% PFA for 20 min, in ice-cold methanol for 10 min (for γ-tubulin and centrin antibodies) or for 10 min in 1× PTEMF extraction buffer (20 mM PIPES, pH 6.8, 10 mM EGTA, 1 mM MgCl 2, 0.2% Triton X-100 and 4% PFA) prepared fresh from 4× stock solution (for all the mitotic checkpoint antibodies). Cells were permeabilized by 2×3 min wash in 0.1% Triton-X-PBS (PBS-TX), followed by blocking in PBS-TX containing 1% BSA for 30 min at room temperature, before being processed for immunofluorescence. Primary antibodies were diluted in 1% BSA in 1× PBS-TX in a 37°C incubator for 1 hr. Primary antibody dilutions were as follows: mouse anti-α-tubulin (DM1α, Sigma, 1∶1000), rabbit anti-γ-tubulin (Sigma, 1∶1000), rabbit anti-pericentrin (Bethyl, 1∶300), mouse anti-centrin-3 (Abnova, 1∶500), rabbit anti-TPX2 (Bethyl, 1∶100), mouse anti-Hec1 (GeneTex, 1∶100), human anti-centromere antibody (ACA, 1∶2000), mouse anti-BubR1 (Millipore, 1∶50), mouse anti-Mad1 (a kind gift from Dr. Andrea Musacchio, 1∶300), sheep anti-Mad2 (a kind gift from Dr. Steven Taylor's Laboratory, 1∶200), rabbit anti-Zwint-1 (Bethyl, 1∶100). Secondary antibodies conjugated to FITC, TRITC or Cy-5 (all from Jackson Laboratories) were chosen as appropriate and used as recommended by the supplier. DNA was counterstained with DAPI.

Kaczmarczyk A, & Sullivan K.F. (2014). CENP-W Plays a Role in Maintaining Bipolar Spindle Structure. PLoS ONE, 9(10), e106464.

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Immunofluorescence Staining Protocol

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For immunofluorescence, cells were plated on #1.5 25 mm coverslips coated with 1 mg/mL poly-L-lysine. Cells were fixed with 95% methanol + 5 mM EGTA at −20°C for 3 min, washed with TBS-T (0.1% Triton-X-100 in TBS), and blocked with 2% BSA in TBS-T for 1 hr. Primary and secondary antibodies were diluted in TBS-T+2% BSA and incubated with cells overnight at 4°C (primary) or for 20 min at room temperature (secondary). DNA was labeled with Hoescht 33342 (Sigma, St. Louis, MO) before cells were mounted in ProLongGold Antifade (P36934; Thermo Fisher). Cells were imaged using the spinning disk confocal microscope described above. Antibodies: mouse anti-α-tubulin DM1α (T6199; Sigma), rabbit anti-α-tubulin (ab18251; Abcam, Cambridge, UK), rabbit anti-NuMA (NB500-174; Novus Biologicals, Littleton, CO), mouse anti-p150-Glued (610473; BD Biosciences, San Jose, CA), mouse anti-α-tubulin DM1α conjugated to Alexa488 (8058S; Cell Signaling, Danvers, MA), mouse anti-dynein intermediate chain (MAB1618MI; Millipore, Billerica, MA), rabbit anti-EB1 (sc-15347; Santa Cruz Biotechnology, Dallas, TX), rabbit anti-KANSL1 (PAB20355; Abnova, Taipei City, Taiwan), rabbit anti-CAMSAP1 (NBP1-26645; Novus Biologicals), mouse anti-actin (MAB1501; Millipore), rabbit anti-γ-tubulin (T3559; Sigma), and camel nanobody against GFP coupled to Atto488 (gba-488; ChromoTek, Hauppauge, NY).

Hueschen C.L., Kenny S.J., Xu K, & Dumont S. (2017). NuMA recruits dynein activity to microtubule minus-ends at mitosis. eLife, 6, e29328.

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Immunofluorescence of Ablated Cells

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For immunofluorescence of individual cells after ablation, cells were live imaged on coverslips photoetched with a labeled grid (Thermo Fisher Scientific). After ablation, cells were fixed in 95% methanol with 5 mM EGTA for 3 min. The time between laser ablation and fixation was usually ∼30 s, but could be as fast as 15 s. The following antibodies and dyes were used: mouse anti–α-tubulin DM1α (1:1,000; Sigma-Aldrich), rabbit anti-NuMA (1:300; Novus Biologicals), mouse anti–p150-Glued (1:500; BD), human anti-centromere protein (CREST; 1:25; Antibodies, Inc.), mouse anti–α-tubulin DM1α conjugated to Alexa 488 (1:50; Cell Signaling Technology), fluorescent secondary antibodies (1:500; Invitrogen), and Hoescht 33342 (Sigma-Aldrich). After staining, we identified the ablated cell using the coverslip grid.

Elting M.W., Hueschen C.L., Udy D.B, & Dumont S. (2014). Force on spindle microtubule minus ends moves chromosomes. The Journal of Cell Biology, 206(2), 245-256.

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Antibodies Used for Western Blot and Immunofluorescence

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Mouse anti-GFP (11 814 460 001; Roche), rabbit anti-GTSE1 (custom generated; described in Scolz et al [2012] (link)), and rabbit anti-Aurora kinase B (ab2254; Abcam) were used for Western blots. Mouse anti–α-tubulin (DM1α; Sigma-Aldrich), rabbit anti-Cep135 (custom generated, described in Bird and Hyman [2008] (link)), goat anti-GFP (MPI Dresden; described in Poser et al [2008] (link)), and goat anti-Aurora A (sc-27883; Santa Cruz) were used for both Western blot and immunofluorescence. Donkey anti-goat Alexa 488 (705 545 147; Jackson Immunoresearch), donkey anti-mouse Alexa 594 (A90-337D4; Bethyl Laboratories), and donkey anti-rabbit Alexa 650 (A120-208D5; Bethyl Laboratories) were used in immunofluorescence. Donkey anti-goat HRP (SC-2020; Santa Cruz), sheep anti-mouse HRP (NXA931-1ml; Amersham), and donkey anti-rabbit HRP (NXA934-1ml; Amersham) were used for Western Blots.

Rondelet A., Pozniakovsky A., Namboodiri D., Cardoso da Silva R., Singh D., Leuschner M., Poser I., Ssykor A., Berlitz J., Schmidt N., Röhder L., Vader G., Hyman A.A, & Bird A.W. (2020). ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes. Life Science Alliance, 4(2), e202000836.

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Immunofluorescence Staining of Cellular Structures

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For immunofluorescence, cells were plated on #1.5 25 mm coverslips coated with 1 mg/mL poly-L-lysine. Cells were fixed with 95% methanol + 5 mM EGTA at −20°C for 3 min, washed with TBS-T (0.1% Triton-X-100 in Tris-buffered saline), and blocked with 2% BSA in TBS-T for 1 h. Primary and secondary antibodies were diluted in TBS-T + 2% BSA and incubated with cells overnight at 4°C (primary) or for 20 min at room temperature (secondary). DNA was labeled with Hoescht 33342 (Sigma) before cells were mounted in ProLongGold Antifade (Thermo Fisher). Cells were imaged using the spinning disk confocal microscope described above. Antibodies: mouse anti-α-tubulin DM1α (T6199; Sigma), rabbit anti-α-tubulin (ab18251; Abcam, Cambridge, UK), rabbit anti-NuMA (NB500–174; Novus Biologicals), mouse anti-α tubulin DM1α conjugated to Alexa488 (8058S; Cell Signaling), mouse anti-dynein intermediate chain (MAB1618MI; Millipore), rabbit anti-γ-tubulin (T3559; Sigma), and mouse anti-centrin (04–1624; Millipore).

Hueschen C.L., Galstyan V., Amouzgar M., Phillips R, & Dumont S. (2019). Microtubule end-clustering maintains a steady-state spindle shape. Current biology : CB, 29(4), 700-708.e5.

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Immunostaining of Drosophila Embryos

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Embryos were fixed in a 1:4 solution of 4% paraformaldehyde: heptane for 20 min and devitellinized in methanol. For visualization of MTs, embryos were prepared as previously described (Theurkauf, 1994 (link)). Briefly, embryos were fixed in a 1:1 mixture of 37% paraformaldehyde: heptane for 3 min, rinsed in PBS, and manually devitellinized using 30G PrecisionGlide needles (BD). Embryos were blocked in BBT buffer (PBS supplemented with 0.1% BSA and 0.1% Tween-20); for Asl staining, 0.5% BSA was used. Samples were incubated overnight at 4°C with primary antibody in BBT, further blocked in BBT supplemented with 2% normal goat serum, and incubated for 2 hr at room temperature with secondary antibody with DAPI. Embryos were mounted in Aqua-Poly/Mount (Polysciences, Inc.) prior to imaging.
The following primary antibodies were used: rabbit anti-Vas (1:2000, gift from P. Lasko, McGill University), rat anti-Vas (1:10, DSHB), guinea pig anti-Asl (1:4000, gift from G. Rogers, University of Arizona), mouse anti-α-Tubulin DM1α (1:500, Sigma-Aldrich T6199), rabbit anti-phospho-Histone H3 Ser10 (pH3; 1:1000, Millipore 05–570), mouse anti-phospho-Tyrosine 4G10 (pTyr; 1:1000, Millipore 05–321), Alexa 568 conjugated phalloidin (Invitrogen #12380). Secondary antibodies: Alexa Fluor 488, 568, or 647 (1:500, Molecular Probes), and DAPI (10 ng/ml, ThermoFisher).

Fang J, & Lerit D.A. (2019). Drosophila pericentrin-like protein promotes the formation of primordial germ cells. Genesis (New York, N.Y. : 2000), 58(3-4), e23347.

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Immunostaining of Neuronal Cytoskeleton Proteins

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See also the Keynote Resources Table. Neurons were immunostained with the following antibodies: mouse anti-α-tubulin (DM1α, 1:500; SIGMA), rabbit anti-SEPT7 (1:500; IBL America), rabbit anti-Myosin IIB (1:500, Covance), rabbit anti-Arp2 (1:100, ECM Biosciences), chicken anti-MAP2 (1:2000; EMD Millipore), mouse anti-Tau1 (1:500, EMD Millipore), mouse anti-GM130 (Golgi, 1:200, BD Transduction), mouse anti-Ankryn 3 (1:100, Novus Biologicals), mouse anti-SEPT5 (1:500, Santa Cruz), rabbit anti-SEPT11 (1:500, Millipore), mouse anti-TUJ1 (Proteintech). F(ab’)₂ fragment affinity-purified secondary antibodies (1:200) were purchased from Jackson ImmunoResearch Laboratories and included donkey anti-mouse, -rabbit, and -chicken antibodies conjugated with AMCA, Alexa488, Alexa594 or Alexa647. To co-stain for Sept7 and MyoIIB and Arp2, rabbit anti-MyoIIB or Arp2 primary antibody was conjugated with anti-rabbit Alexa Fluor 594 using the Zenon Rabbit IgG Labeling Kit (Thermo Fisher Scientific). To stain actin, phalloidin conjugated with iFluor 647 (1:200, Abcam) was used.

Radler M.R., Liu X., Peng M., Doyle B., Toyo-Oka K, & Spiliotis E.T. (2022). Pyramidal neuron morphogenesis requires a septin network that stabilizes filopodia and suppresses lamellipodia during neurite initiation. Current biology : CB, 33(3), 434-448.e8.

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