Mouse α tubulin

Manufactured by Merck Group

Sourced in United States

Mouse α-tubulin is a protein component of microtubules, which are cytoskeletal structures found in eukaryotic cells. It plays a crucial role in the formation and function of the cytoskeleton.

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Lab products found in correlation

Enhanced chemi luminescence (ecl), by GE Healthcare (3 mentions) Rat α ollas l2, by Novus Biologicals (2 mentions) Odyssey fc imager, by LI COR (2 mentions) Rabbit α actin, by Merck Group (2 mentions) α rabbit hrp, by Cell Signaling Technology (2 mentions) Odyssey classic scanner, by LI COR (2 mentions) Goat α mouse 800, by LI COR (2 mentions) Hrp coupled secondary antibody, by Merck Group (2 mentions) Fusion sl 3500 wl system, by Avantor (2 mentions) Cryoprep dry impactor, by Covaris (2 mentions) Proteinase inhibitor, by Thermo Fisher Scientific (2 mentions) Mouse anti cas9, by Active Motif (2 mentions) Chicken α gfp, by Abcam (2 mentions) Odyssey imaging system, by LI COR (2 mentions) Smartpool reagents, by Horizon Discovery (1 mentions) T9026, by Merck Group (1 mentions) Goat α mouse irdye 680, by LI COR (1 mentions) Mini protean 3 system, by Bio-Rad (1 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Bca assay kit, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

α-tubulin (1242 citations) Mouse anti-α-tubulin DM1A (19 citations) Mouse anti-α-tubulin (T5168, (13 citations) Mouse anti–α-tubulin monoclonal DM1A (4 citations) Mouse antibody against α-tubulin (4 citations) Mouse α-tubulin (clone B-5-1-2, (3 citations) Acetylated α‐tubulin mouse IgG (clone 6‐11 B‐1; (2 citations) Mouse monoclonal anti-α-tubulin antibody (DM1A; (2 citations) Mouse anti-α-tubulin (T6199) (2 citations) Mouse anti-acetylated-α-tubulin clone 6-11B-1 (2 citations)

32 protocols using mouse α tubulin

Protein Detection via Western Blotting

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Western blots were performed by running worm lysates on 7% Tris Acetate SDS PAGE precast gels (Bio-Rad Hercules, CA). Protein was transferred to nitrocellulose membrane which was pre-blocked in 5% Milk diluted in PBS-Tween (0.1%) for 5 min (three times). The membrane was then incubated with Primary antibody for at least 18 hr at 4° C or 2 hr at room temperature. Membranes were washed and blocked in 5% milk for 5 min (three times) and incubated with secondary HRP conjugated antibody for 45 min at room temperature. Membranes were washed in 5% milk for 5 min (two times) and PBST for 5 min (one time). The membranes were then exposed to ECL substrate for 1 min and then exposed to film. Primary antibody dilutions (in 5% Milk PBST): Rat α OLLAS-L2 (1:1000, Novus Biological Littleton, CO), Mouse α Tubulin (1:1000, Sigma St. Louis, MO).

Smith J., Calidas D., Schmidt H., Lu T., Rasoloson D, & Seydoux G. (2016). Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3. eLife, 5, e21337.

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Silencing Gal-1 in Cancer Cells

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PANC-1 cells were transduced with shGal1 or shCtl by lentiviral infection as described in Supplementary Material. F88.2 and HPSE cells were transfected with 50nM of Gal-1 siRNA or an irrelevant siRNA (SMARTpool® Reagents, Dharmacon). To control knockdown efficiency, cells were directly lysed 72 h after transfection and protein levels measured by Western blot (WB) using Rabbit α-Gal-1 (Sigma) or mouse α-Tubulin (Sigma), peroxidase-conjugated secondary antibodies and ECL (GE Healthcare).

Martínez-Bosch N., Fernández-Barrena M.G., Moreno M., Ortiz-Zapater E., André S., Gabius H.J., Hwang R.F., Poirier F., Munné-Collado J., Iglesias M., Navas C., Guerra C., Fernández-Zapico M.E, & Navarro P. (2014). Galectin-1 drives pancreatic carcinogenesis through stroma remodeling and Hedgehog signaling activation. Cancer research, 74(13), 3512-3524.

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Western Blot Antibody Validation

Cited 1 time

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Western blot assays were performed as described previously (46 (link)), using primary antibodies rabbit-α-puromycin (MABE343, 1:1,000; Merck Millipore), rabbit-α-eIF2α (9722, 1:2,000; Cell Signaling), rabbit-α-eIF2α-P (ab32157, 1:1,000; Abcam), or mouse-α-tubulin (T9026, 1:5,000; Sigma-Aldrich), and secondary antibodies goat-α-mouse-IRDye680 (1:15,000; LI-COR) or goat-α-rabbit-IRDye800 (1:15,000; LI-COR).

Rabouw H.H., Langereis M.A., Anand A.A., Visser L.J., de Groot R.J., Walter P, & van Kuppeveld F.J. (2019). Small molecule ISRIB suppresses the integrated stress response within a defined window of activation. Proceedings of the National Academy of Sciences of the United States of America, 116(6), 2097-2102.

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Western Blot Analysis of Cellular Proteins

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Tissue lysates were homogenized, lysed, and quantified as above, and then treated with an equal amount of SDS-PAGE loading buffer (62.5 mM Tris-Cl, pH 6.8, 20% glycerol, 7.5% SDS, 5% 2-mercaptoethanol, and 250 mM DTT) and denatured by heating 95°C. Proteins were then separated using an SDS-PAGE gel and transferred to a polyvinylidene Immobilon-FL membrane. Membranes were washed in PBS, then soaked in 5% milk + PBST to block nonspecific binding. The membranes were incubated in a primary rabbit α-human A3A/B/G antibody 1:1,000 (Brown et al.) and mouse α-tubulin 1:10,000 (Sigma Aldrich), or rabbit α-actin 1:5,000 (Sigma Aldrich) at 4°C overnight. Membranes were then washed in PBST six times for 5 minutes each, then incubated for one hour with secondary α-rabbit HRP (Cell Signaling Technology) and goat α-mouse 800 (LI-COR) at 1:10,000 dilutions supplemented with 0.02% SDS. These membranes were then washed 5 times in PBST and one time in PBS for 5 minutes each, then imaged using an Odyssey Classic scanner and Odyssey Fc imager (LI-COR)

Durfee C., Temiz N.A., Levin-Klein R., Argyris P.P., Alsøe L., Carracedo S., de la Vega A.A., Proehl J., Holzhauer A.M., Seeman Z.J., Lin Y.H., Vogel R.I., Sotillo R., Nilsen H, & Harris R.S. (2023). Human APOBEC3B promotes tumor heterogeneity in vivo including signature mutations and metastases. bioRxiv.

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Western Blot Protein Quantification

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To determine protein concentration, a BCA assay kit (Thermo Scientific) was used with a microplate reader reading of absorbable at 562 nm. After protein lysates were denatured in Laemmli buffer for 5 min at 95 °C, 50 µg of supernatant protein and 25 µg of pellet protein were loaded on a 12% polyacrylamide gel. A discontinuous Laemmli SDS-PAGE was used with the Bio-Rad mini Protean 3 system and proteins were transferred onto nitrocellulose membrane (Enhanced chemiluminescence, ECL, 0.2 µm pore size, Amersham). To block unspecific binding, membranes were incubated in 5% (w/v) low-fat milk powder in Tris-buffered saline supplemented with 0.1% (v/v) tween (TBST). Primary antibodies were incubated in blocking solution at 4 °C overnight: rabbit α-LC3 (1:1000, MBL, PD014), mouse α-p62 (1:1000, Abnova, H00008878-M01), mouse α-Tubulin (1:10000, Sigma, T9026). HRP-conjugated α-mouse and α-rabbit secondary antibodies (Calbiochem) were applied for 1 hour at room temperature in blocking solution. SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and the ChemiDoc™ XRS+ System were used to visualize the protein of interest. Densiometric analysis was performed using the Image Studio Lite software.

Baron O., Grieshober D., Dias C, & Fanto M. (2019). Fat cadherins in mouse models of degenerative ataxias. Scientific Reports, 9, 16155.

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Fly Head Protein Extraction and Western Blot

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Frozen female flies (1 d, 10 d) were decapitated by vigorous shaking, and heads were separated by passing through a metal sieve. Approximately 85-100 frozen heads were pulverized by Covaris CryoPREP® Dry Impactor, the powder was suspended in 100 μL 1x LSB (0.0625 M Tris; 2% SDS, 10% glycerol, 5% 2-mercapto-ethanol) and sonicated in a Bioruptor instrument for 30 cycles of 30 s ON, 30 s OFF at 4°C. Extracts were centrifuged at 16,000 g for 20 min at RT, the supernatant was transferred to a new tube avoiding the lipid layer on top and centrifugation was repeated twice. The remaining supernatant was heated at 95°C for 5 minutes, loaded onto 12% or 8% SDS-PAGE gels and blotted to nitrocellulose membrane. Membranes were blocked in 5% milk/PBS and incubated overnight at 4°C with primary antibodies followed by washing in PBS and incubation with HRP-coupled secondary antibody (Sigma, 1:10,000). Signal development was done by incubation with ECL (GE Healthcare) followed by detection using a Fusion-SL 3500-WL system (PeqLab) and Fusion software (v15.17). The following antibodies were used: rabbit α-dmXNP15879 (1:1,000; [Emelyanov et al., 2010 (link)]; rabbit α-dmHIRA (1:5,000), rabbit α-dmASF1 (1:20,000; kind gift of Jessica Tyler); mouse α-tubulin (1:10,000; Sigma T5168).

Schoberleitner I., Bauer I., Huang A., Andreyeva E.N., Sebald J., Pascher K., Rieder D., Brunner M., Podhraski V., Oemer G., Cázarez-García D., Rieder L., Keller M.A., Winkler R., Fyodorov D.V, & Lusser A. (2021). CHD1 controls H3.3 incorporation in adult brain chromatin to maintain metabolic homeostasis and normal lifespan. Cell reports, 37(1), 109769.

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Molecular Biology Reagents and Antibodies Used in Malaria Research

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The molecular biology reagents were purchased from either MilliporeSigma, USA or Thermo Fisher Scientific, USA, unless otherwise stated. All the restriction enzymes and DNA polymerases were purchased from New England Biolabs, and all the oligonucleotides were purchased from Integrated DNA Technologies, Inc., USA. The following primary antibodies, antisera, and dilutions were utilized: mouse α-tubulin (1:250; Sigma-Aldrich; catalog# T5168), rabbit α-Pfg377 (1:250; kindly gifted by Prof. Pietro Alano at Istituto Superiore di Sanità, Rome, Italy) (29 (link)), and mouse α-PfP230p (1:200, kindly gifted by Prof. Kim C. Williamson, Uniformed Services University of the Health Sciences, USA (28 (link)). Reagents obtained through BEI Resources, NIAID, and NIH include: hybridoma 4B7 α-Pfs25-kilodalton gamete surface protein (Pfs25), MRA-315, contributed by Louis H. Miller and Allan Saul (1:1 in 3% bovine serum albumin/phosphate-buffered saline, mouse). The Alexa Fluor-conjugated secondary antibodies utilized for IFAs were procured from Thermo Fisher Scientific, USA.

Mauer S., Camargo N., Abatiyow B.A., Gargaro O.R., Kappe S.H, & Kumar S. (2023). Plasmodium microtubule-binding protein EB1 is critical for partitioning of nuclei in male gametogenesis. mBio, 14(4), e00822-23.

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Protein Expression Analysis Protocol

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For Western blot, cells were lyzed in RIPA buffer (50 mM Tris–Cl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% Na deoxycholate, 0.1% SDS, 1 mM EDTA). Proteins were resolved by SDS-PAGE and then transferred onto PVDF membranes (Millipore). All buffers contained a cocktail of protease inhibitors (Boehringer, Ingelheim am Rhein, Germany). Membranes were developed using the enhanced chemiluminescence (ECL westar, Cynagen, Bologna, Italy). Bands were analyzed by chemiluminescence imaging system, Alliance 2.7 (UVITEC, Cambridge, UK) and quantified by the software Alliance V_1607.
The following primary antibodies were used: rabbit α-PTTG1 (Abcam), mouse α-MMP-2 (Thermo Fisher, Waltham, MA, USA), mouse α-HA (BioLegend, San Diego, CA, USA), mouse α-tubulin (Sigma, St. Louis, MO, USA), mouse α-actin monoclonal antibody C-40 (Sigma), and rabbit α-Sp1 (Santa Cruz, Santa Cruz, CA, USA).

Teveroni E., Di Nicuolo F., Bianchetti G., Epstein A.L., Grande G., Maulucci G., De Spirito M., Pontecorvi A., Milardi D, & Mancini F. (2021). Nuclear Localization of PTTG1 Promotes Migration and Invasion of Seminoma Tumor through Activation of MMP-2. Cancers, 13(2), 212.

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Protein Expression Analysis Protocol

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Cells were lysed by RIPA buffer with proteinase inhibitor (Invitrogen), and subject to standard immunoblotting analysis. Mouse anti-Cas9 (1:1000, Active Motif), mouse α-Tubulin (1:1000, Sigma), mouse anti-FMR1polyG (1:1000, EMD Millipore), rabbit anti-FMRP (1:100, Cell Signaling) antibodies were used.

Liu X.S., Wu H., Krzisch M., Wu X., Graef J., Muffat J., Hnisz D., Li C.H., Yuan B., Xu C., Li Y., Vershkov D., Cacace A., Young R.A, & Jaenisch R. (2018). Rescue of Fragile X syndrome neurons by DNA methylation editing of the FMR1 gene. Cell, 172(5), 979-992.e6.

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Western Blot Analysis of Protein Samples

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Protein samples were separated on 10–12% SDS-PAGE gels and blotted to nitrocellulose membrane. Antibody incubation and detection were performed as described in Supplementary Information. The following antibodies were used: mouse α-Flag (1:5000; Proteintech 20543-1-AP), rabbit α Drosophila CENP-A (1:2000; Abcam ab10887), rabbit α-H3 (1:20 000; Millipore 07-690), mouse α-tubulin (1:10 000; Sigma T5168), rabbit α-CENP-A-S20p (1:1000; this study). Secondary anti-rabbit and anti-mouse IgG antibodies (Sigma) coupled to horseradish peroxidase (HRP) were used at 1:10 000. Raw images of western blots from Figures 1E and 4C are shown in Supplementary Figures S10 and S11.

Huang A., Kremser L., Schuler F., Wilflingseder D., Lindner H., Geley S, & Lusser A. (2019). Phosphorylation of Drosophila CENP-A on serine 20 regulates protein turn-over and centromere-specific loading. Nucleic Acids Research, 47(20), 10754-10770.

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