Mouse anti acetylated α tubulin

Manufactured by Merck Group

Sourced in United States

The Mouse anti-acetylated α-tubulin is a primary antibody that specifically binds to the acetylated form of the alpha-tubulin protein. Alpha-tubulin is a key component of the cytoskeleton in eukaryotic cells. Acetylation of alpha-tubulin is a post-translational modification that can influence the stability and function of microtubules.

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Lab products found in correlation

Fluoro gel, by Electron Microscopy Sciences (7 mentions) Phalloidin 650, by Thermo Fisher Scientific (6 mentions) Mouse anti flag m2, by Merck Group (6 mentions) Alexa fluor plus 405 phalloidin, by Thermo Fisher Scientific (4 mentions) Mouse anti α tubulin, by Merck Group (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Rabbit anti arl13b, by Proteintech (3 mentions) Rabbit anti dykddddk, by Cell Signaling Technology (3 mentions) Alexa fluor 594 donkey anti mouse, by Thermo Fisher Scientific (3 mentions) Rabbit anti γ tubulin, by Merck Group (2 mentions) Nitrocellulose membrane, by LI COR (2 mentions) Odyssey clx imaging system, by LI COR (2 mentions) Rabbit anti hmox 1 antibody, by Cell Signaling Technology (2 mentions) Mouse anti involucrin, by Thermo Fisher Scientific (2 mentions) Irdye conjugated secondary antibody, by LI COR (2 mentions) Image studio software, by LI COR (2 mentions) Cy 2 or cy 5 conjugated goat anti mouse secondary antibodies, by Thermo Fisher Scientific (2 mentions) Phalloidin 568 or 647, by Thermo Fisher Scientific (2 mentions) Rabbit anti raptor, by Merck Group (2 mentions) Hpa038941, by Merck Group (2 mentions)

Close spelling variants of this product

Mouse anti-acetylated α-tubulin (6-11B-1) (4 citations) Mouse anti-acetylated-α-tubulin clone 6-11B-1 (2 citations) Mouse monoclonal anti-acetylated α-tubulin (8 citations) Mouse anti‐acetylated α‐tubulin antibody (7 citations) Mouse anti-acetylated α-tubulin monoclonal antibody (2 citations) Mouse monoclonal anti-acetylated α-tubulin clone 6-11B-1 (2 citations) Anti-acetylated α-tubulin (46 citations) Mouse anti-acetylated tubulin (92 citations) Acetylated α-tubulin (67 citations) Mouse anti-α-tubulin (406 citations)

39 protocols using mouse anti acetylated α tubulin

Immunostaining and Microtubule Regrowth Assay in HeLa Cells

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HeLa cells were cultured on ethanol-sterilised coverslips in 12-well plates and synchronised prior to fixation with ice-cold methanol at −20 °C for 10 min. Fixed cells were permeabilised with 0.3% Triton X-100 in 1 × PBS for 15 min and blocked with 2% BSA for 30 min at room temperature. Mouse anti-α-tubulin 1:2000 (Sigma), mouse anti-acetylated α-tubulin 1:200 (Sigma), rabbit anti-γ-tubulin 1:200 (Sigma), and rabbit anti-MCAK (Cytoskeleton) were used to stain the respective proteins. DNA was stained with Hoechst 33342 (Invitrogen). Confocal images were collected using the UltraVIEW Vox Spinning disc confocal system (PerkinElmer). Images were processed using Volocity^TM software (PerkinElmer) or Image J (National Institutes of Health, Bethesda, MD). STED imaging was conducted with the STED TCS SP8 system (Leica Microsystems) and both the STED and confocal images were deconvoluted using Huygens Titan (Scientific Volume Imaging). For microtubule regrowth assays, synchronized HeLa cells were washed with cold medium and placed on ice for 30 min before changing into prewarmed medium. The cells were fixed and immunostained at the indicated time points after medium replacement to monitor microtubule regrowth57 (link).

Li C., Zhang Y., Yang Q., Ye F., Sun S.Y., Chen E.S, & Liou Y.C. (2016). NuSAP modulates the dynamics of kinetochore microtubules by attenuating MCAK depolymerisation activity. Scientific Reports, 6, 18773.

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Immunofluorescence Staining of Acetylated α-Tubulin

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Cells were seeded and cultured on coverslips. After treatment for the indicated time, cells were fixed in 4% paraformaldehyde for 15 min and incubated in blocking solution (10% donkey serum and 0.1% Triton X-100 in PBS) for 1-2 hours at room temperature. Cells were then incubated with primary antibody (mouse anti-acetylated α-tubulin; Sigma, USA) overnight at 4°C in the cold room followed by the secondary antibody (Alexa Fluor 488 AffiniPure Donkey Anti-Mouse IgG; Jackson ImmunoResearch Laboratories, USA) for 2 hours at room temperature under dark conditions. Finally, cells were stained with DAPI (4′,6-diamidino-2-phenylindole) and mounted with antifluorescence quencher (Promotor, China). Fluorescence images were directly taken using an inverted confocal microscope (Olympus, Japan).

Liu L., Sheng J.Q., Wang M.R., Gan Y., Wu X.L., Liao J.Z., Tian D.A., He X.X, & Li P.Y. (2019). Primary Cilia Blockage Promotes the Malignant Behaviors of Hepatocellular Carcinoma via Induction of Autophagy. BioMed Research International, 2019, 5202750.

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Immunostaining of Cilia and Microtubules

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Transfected cells were fixed in 100% methanol at −20°C for 10 min. After washing with 1× PBS, the cells were blocked with blocking solution (4% donkey serum in PBST) at RT for 1 h. Cells were incubated with primary antibodies: mouse anti‐acetylated α tubulin (Sigma, T7451, MO, USA, 1:2,000), mouse anti‐GT335 (Adipogen, AG‐20B‐0020, CA, USA, 1:2,000), rabbit anti‐γ tubulin (Santa Cruz, SC‐10732, CA, USA 1:200), rabbit anti‐ARL13b (Proteintech, 17711‐1‐AP, IL, USA, 1:1,000) antibodies at 4°C overnight in blocking solution. After washing with 1× PBS, the cells were incubated with Alexa Flour^® 488‐conjugated secondary antibodies (Invitrogen, mouse A11001, rabbit A11008, 1:500) or Alexa Flour^® 594‐conjugated secondary antibodies (Invitrogen, mouse A11005, rabbit A11012, 1:500) at RT for 1 h. Randomly selected fields (≥ 5) were photographed using the confocal microscope (Carl Zeiss, LSM 700 or LSM 780, Germany) in all of imaging analyses. The intensity and length of cilia were measured using ImageJ software (NIH, USA).

Ki S.M., Kim J.H., Won S.Y., Oh S.J., Lee I.Y., Bae Y., Chung K.W., Choi B., Park B., Choi E, & Lee J.E. (2019). CEP41‐mediated ciliary tubulin glutamylation drives angiogenesis through AURKA‐dependent deciliation. EMBO Reports, 21(2), e48290.

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Immunoblotting of Cell Lysates

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Immunoblotting was conducted as described previously [34 (link)]. Briefly, cell lysates extracted using the Pierce M-PER Mammalian Cell Lysis Buffer supplemented with 1% SIGMAFAST^™ Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA) were denatured, separated on a 4–12% NuPage^® Novex^® Bis-Tris gradient gel (Life Technologies, Carlsbad, CA, USA) at 200 V for 45 min, and transferred onto a nitrocellulose membrane (LI-COR, Lincoln, NE, USA) at 30 V for 1 h. Proteins were detected by incubating first with primary antibodies (rabbit anti-HMOX-1 antibody, Cell Signaling, Danvers, MA, USA; mouse anti-acetylated α-tubulin, mouse anti-AKR1B10, rabbit-anti-DNAI1, and mouse anti-CDC20B, Sigma–Aldrich; mouse anti-CK13 and goat anti-MGMT, Santa Cruz, Dallas, TX, USA; mouse anti-involucrin, Thermo Fisher Scientific, Waltham, MA, USA; rabbit anti-ADH1C and rabbit anti-FANCD2, Abcam, Cambridge, MA, USA), followed by a 30-min incubation with the IRDye-conjugated secondary antibodies (LI-COR). Images were taken using an Odyssey^® CLx Imaging System (LI-COR). Densitometry was conducted using LI-COR Image Studio software.

Ren B., Wu Q., Muskhelishvili L., Davis K., Wang Y., Rua D, & Cao X. (2022). Evaluating the Sub-Acute Toxicity of Formaldehyde Fumes in an In Vitro Human Airway Epithelial Tissue Model. International Journal of Molecular Sciences, 23(5), 2593.

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Cilia Visualization in Embryos

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Embryos were fixed with 3% PFA/PBS and blocked in 10% heat-inactivated goal serum (HIGS)/PBS after washing with PBS. To visualize cilia, embryos were incubated with mouse anti-acetylated α-tubulin (T7451; Sigma-Aldrich, 1:500) in 5% HIGS/PBS for an hour at room temperature. Then, Cy-2- or Cy-5-conjugated goat anti-mouse secondary antibodies (Thermo Fisher Scientific) were used at a 1:750 dilution in 5% HIGS/PBS. To visualize actin and nuceli, Phalloidin 650 (PI21838, Invitrogen, 1:300) and DAPI (#62248, Thermo Fisher Scientific, 1:300) were used. Following staining, embryo tails were mounted between two coverslips as previously described (Werner and Mitchell, 2013 ) using Fluoro-Gel (#1798510, Electron Microscopy Sciences).

Ventrella R., Kim S.K., Sheridan J., Grata A., Bresteau E., Hassan O., Suva E.E., Walentek P, & Mitchell B. (2023). Bidirectional multiciliated cell extrusion is controlled by Notch driven basal extrusion and Piezo 1 driven apical extrusion. bioRxiv.

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Embryo and RPE Cell Immunostaining Protocol

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For most stainings, embryos were fixed with 3% PFA in 80 mM K⁺ Pipes, pH 6.8, containing 2 mM MgCl₂ and 5 mM EDTA for 2 h (Werner and Mitchell, 2013 (link)). For AE1, acetylated tubulin, γ-tubulin, and CLAMP mAb staining, embryos were fixed in 100% ice-cold methanol followed by rehydration in EtOH. RPE cells were fixed with 3% PFA in 80 mM K⁺ Pipes, pH 6.8, containing 2 mM MgCl₂ and 5 mM EDTA for 10 min or 100% ice-cold methanol for 20 min. RPE cells and embryos were blocked with 5% normal donkey serum in PBS with 0.1% Triton X-100 for 1 h. The following primary antibodies were used according to the manufacturer’s recommended dilutions: mouse anti–β-tubulin (7–10) and mouse anti-AE1 (IVF12; both from the Developmental Studies Hybridoma Bank), mouse anti-acetylated α-tubulin (T7451; Sigma-Aldrich), mouse anti–γ-tubulin (GTU-88; Sigma-Aldrich), rabbit anti–ZO-1 (61-7300; Invitrogen), and Cy-2–, Cy-3–, or Cy-5–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). Phalloidin 568 or 647 (Invitrogen) were used to visualize actin. Embryos were mounted between two coverslips using Fluoro-Gel (Electron Microscopy Sciences; Werner and Mitchell, 2013 (link)).

Werner M.E., Mitchell J.W., Putzbach W., Bacon E., Kim S.K, & Mitchell B.J. (2014). Radial intercalation is regulated by the Par complex and the microtubule-stabilizing protein CLAMP/Spef1. The Journal of Cell Biology, 206(3), 367-376.

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Immunofluorescence Staining of Embryos

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For antibody staining, embryos were fixed with 3% PFA in PBS, blocked in 10% goat serum, and primary and secondary antibody solutions were prepared in 5% goat serum. Mouse anti-acetylated α-tubulin (T7451; Sigma-Aldrich) was used at a 1:500 dilution, mouse anti-beta tubulin (DHSB; E7) supernatant was used at a 1:10 dilution. Mouse anti-FLAG M2 (F3165; Sigma) and rabbit anti-DYKDDDDK (2368; Cell Signaling) were used at 1:100 dilutions. E7 (anti-tubulin) was deposited to the DSHB by Klymkowsky, M (Chu and Klymkowsky, 1989 (link)). Cy-2–, Cy-3–, or Cy-5–conjugated goat anti-mouse secondary antibodies were used at the manufacturers’ recommended dilution. Phalloidin 650 (1:600, Invitrogen) and Alexa Fluor Plus 405 Phalloidin (1:100, Invitrogen) were used to visualize actin. Embryos were mounted between two coverslips using Fluoro-Gel (Electron Microscopy Sciences).

Collins C., Kim S.K., Ventrella R., Carruzzo H.M., Wortman J.C., Han H., Suva E.E., Mitchell J.W., Yu C.C, & Mitchell B.J. (2021). Tubulin acetylation promotes penetrative capacity of cells undergoing radial intercalation. Cell reports, 36(7), 109556.

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Immunofluorescence Staining of Kidney Proteins

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The dilution and sources for primary antibodies used were as follows: rabbit anti-Vangl1 (1:200, Sigma), rabbit anti-Vangl2 (1:200, Santa Cruz), goat anti-Frizzled6 (1:200, R&D systems), goat anti-Frizzled3 (1:100, R&D systems), rat anti-Frizzled3 (1:100, R&D systems), rat anti-E-Cadherin (1:200, Invitrogen) and mouse anti-E-cadherin (1:200, BD Transduction Laboratories), mouse anti-acetylated-α-tubulin (1:500, Sigma), rabbit anti-phospho-Histone H3 (Ser10) (1:200, Millipore) and mouse anti-β-catenin (1:100, BD Transduction Laboratories^™). The following secondary antibodies were used at 1:500 and purchased from Molecular Probes (now ThermoFisher): Alexa 488, 594 donkey-anti-rabbit, Alexa 488, 546 donkey-anti-goat, Alexa 594, 633 donkey-anti-rat, Alexa 647 chicken-anti-rat and Streptavidin Alexa 594 conjugate. Phalloidin conjugated to Alexa 633 was used at 1:50. Rhodamine-conjugated Dolichos Biflorus Agglutinin lectin (DBA, 1:50, Vector Laboratories) and goat anti-AQP2 (1:200, Santa Cruz) were used to stain collecting duct. Fluorescein-conjugated Lotus Tetragonolobus lectin (LTL, 1:200, Vector Laboratories) and biotinylated Lotus Tetragonolobus lectin (1:200, Vector Laboratories) were used to stain proximal tubules. DAPI (300nM, Sigma), Sytox Green (1:20,000, Invitrogen) and 7-aminoactinomycin D (7-AAD, 1:40, Invitrogen) were used to stain nuclei.

Kunimoto K., Bayly R.D., Vladar E.K., Vonderfecht T., Gallagher A.R, & Axelrod J.D. (2017). Disruption of core planar cell polarity signaling regulates renal tubule morphogenesis but is not cystogenic. Current biology : CB, 27(20), 3120-3131.e4.

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Immunostaining of Mouse Testicular Tissue

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Frozen and paraffin sections of mouse testes were prepared as described (Nagy et al., 2003 ). Dry-down slides of sperm were also used. Samples were fixed in 4% paraformaldehyde for 10 minutes, permeabilized in 0.5% Triton X-100/PBS for 15 minutes and incubated for 1–2 hours in blocking solution (3% BSA and 0.02% Triton X-100 in PBS). Standard procedures were used for immunostaining (Ausubel et al., 2003 ). Primary antibodies used included mouse anti-acetylated-α-tubulin (Sigma, 1:500), chick anti-GFP (Abcam, 1:500), rabbit anti-STK36 (Proteintech, 1:50) and rabbit anti-TEX14 (Abcam, 1:400). Secondary antibodies used included donkey anti-mouse Alexa Fluor 594 (Life Technologies, 1:2000), donkey anti-mouse Alexa Fluor 488 (Life Technologies, 1:2000), donkey anti-rabbit Alexa Fluor 594 (Life Technologies, 1:2000) and donkey anti-chicken Alexa Fluor 488 (Life Technologies, 1:2000).
Confocal images were acquired with a Leica TCS SPE laser scanning confocal system. Confocal stacks were collected using a 0.25 µm-0.5 µm step size along the z-axis. NIH ImageJ was used for image analysis.

Nozawa Y.I., Yao E., Gacayan R., Xu S.M, & Chuang P.T. (2014). Mammalian Fused is essential for sperm head shaping and periaxonemal structure formation during spermatogenesis. Developmental biology, 388(2), 170-180.

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Immunoblotting Analysis of NEK10 Expression

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Protein lysates were prepared in lysis buffer containing 1% Triton X-100, 10 mM β-glycerol phosphate, 10 mM pyrophosphate, 40 mM HEPES pH 7.4, 2.5 mM MgCl₂, and one mini tablet of EDTA-free protease inhibitor (Roche) per 10mL. Lysates were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes before immunoblotting with the indicated antibodies. Primary antibodies and working dilutions were as follows: rabbit anti-NEK10 (Sigma HPA038941, lot R35857, 1:1000), mouse anti-NEK10 (Sigma WH0152110M1, lot 09058–1C9, 1:1000), rabbit anti-GAPDH (Abcam ab9485, 1:2500), mouse anti-FLAG M2 (Sigma F1804, lot SLBS3530V, 1:1000), rabbit anti-Raptor (Millipore 09–217, lot 3236353, 1:1000), mouse anti-β-actin (Santa Cruz sc-47778, lot K1718, 1:1000), mouse anti-acetylated α-tubulin (Sigma T7451, 1:1000). Secondary antibodies and dilutions were as follows: HRP-conjugated anti-rabbit IgG (Cell Signaling Technologies #7074, 1:3000), HRP-conjugated anti-mouse IgG (Cell Signaling Technologies #7076, 1:3000).

Chivukula R.R., Montoro D.T., Leung H.M., Yang J., Shamseldin H.E., Taylor M.S., Dougherty G.W., Zariwala M.A., Carson J., Daniels L.A., Sears P., Black K.E., Hariri L.P., Almogarri I., Frenkel E.M., Vinarsky V., Omran H., Knowles M.R., Tearney G.J., Alkuraya F.S, & Sabatini D.M. (2020). A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance. Nature medicine, 26(2), 244-251.

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