Custom solutions & partnerships Custom antibody development and commercial partnerships to advance your diagnostic and therapeutic discovery. Keep up to date with the latest events Full event breakdown with abstracts, speakers, registration and more View global event calendar Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control Suitable for: Flow Cyt, ICC/IF, IHC-P, WB Reacts with: Mouse, Rat, Human Isotype: IgG1 Mouse, Rat, HumanPredicted to work with: Chicken, Guinea pig, Hamster, Cow, Dog, Pig, Xenopus laevis, Gerbil, African green monkey WB: HeLa, HEK293, HepG2, Caco2, NIH3T3, PC12 cell lysates.Flow Cyt: methanol fixed/Tween permeabilised HeLa cells.ICC/IF: Caco-2, NIH3T3, and SV40LT-SMC cells.IHC-P: Human colon and rat colon tissues. This antibody clone [DM1A] is manufactured by Abcam.Excellent as a protein loading control antibody. DM1A causes the 10 nm filaments to collapse into large lateral aggregates collecting in the cell periphery or tight juxtanuclear caps. It does not block microtubule assembly. It does not inhibit polymerisation or depolymerisation of platelet tubulin in vitro. It blocks (by 70-80%) the ability of tubulin dimers (with GppNHp bound) to promote a stable inhibition of adenylyl cyclase. See references for further information on the above.If you require this antibody in aparticular buffer formulation or aparticular conjugate for your experiments, pleasecontactorders@abcam.comor you can findfurther informationhere.The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q As Storage instructions Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle. Storage buffer pH: 7.40Preservative: 0.02% Sodium azideConstituents: PBS, 6.97% L-Arginine The Abpromise guarantee Our Abpromise guarantee covers the use of ab7291 in the following tested applications. The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user. Flow Cyt Use 1µg for 106 cells. ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody. IHC-P (2) Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. 1/5000 - 1/10000. Detects a band of approximately 50 kDa (predicted molecular weight: 50 kDa). We recommenddiluting ab7291 to 1:10000 and incubating overnight at 4 C. Works under both reducing and non-reducing conditions. We recommend using 3% BSA as the blocking agent, blocking with milk may cause a reduction in signal intensity. Flow CytUse 1µg for 106 cells. ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody. IHC-PUse a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. WB1/5000 - 1/10000. Detects a band of approximately 50 kDa (predicted molecular weight: 50 kDa). We recommenddiluting ab7291 to 1:10000 and incubating overnight at 4 C. Works under both reducing and non-reducing conditions. We recommend using 3% BSA as the blocking agent, blocking with milk may cause a reduction in signal intensity. Function Tubulin is the major constituent of microtubules. It binds two moles of GTP, one at an exchangeable site on the beta chain and one at a non-exchangeable site on the alpha chain. Post-translationalmodifications Some glutamate residues at the C-terminus are polyglutamylated. This modification occurs exclusively on glutamate residues and results in polyglutamate chains on the gamma-carboxyl group. Also monoglycylated but not polyglycylated due to the absence of functional TTLL10 in human. Monoglycylation is mainly limited to tubulin incorporated into axonemes (cilia and flagella) whereas glutamylation is prevalent in neuronal cells, centrioles, axonemes, and the mitotic spindle. Both modifications can coexist on the same protein on adjacent residues, and lowering glycylation levels increases polyglutamylation, and reciprocally. The precise function of such modifications is still unclear but they regulate the assembly and dynamics of axonemal microtubules.Acetylation of alpha chains at Lys-40 stabilizes microtubules and affects affinity and processivity of microtubule motors. This modification has a role in multiple cellular functions, ranging from cell motility, cell cycle progression or cell differentiation to intracellular trafficking and signaling. All lanes : Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilutionLane 1 : HeLa cell lysateLane 2 : PC12 cell lysateLane 3 : SV40LT-SMC cell lysateLane 4 : NIH/3T3 cell lysateLane 5 : Rat liver tissue lysateLane 6 : Rat heart tissue lysateLysates/proteins at 20 µg per lane.SecondaryAll lanes : Goat anti-Mouse IgG H&L (IRDye 800CW) preadsorbed (ab216772) at 1/20000 dilutionPerformed under reducing conditions.Predicted band size: 50 kDaMerged signal (red and green). Green - ab7291 observed at52 kDa. Red - loading control, ab181602, observed at 38 kDa.All samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. ab7291 and ab181602 (Rabbit antiGAPDH loading control) were incubated overnight at 4 C at1/1,000and 1/20,000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H L (IRDye 800CW) preabsorbedab216772and Goat anti-Rabbit IgG H L (IRDye 680RD) preabsorbedab216777secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging. Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)Image from Dai C et al., PLoS One 10(8), Fig 5C. Doi: 10.1371/journal.pone.0063054. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/ ab7291 staining alpha tubulin in human breast cancer cell line by ICC/IF (Immunocytochemistry/Immunofluorescence). Cells were fixed 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS and blocked with 2% bovine serum albumin in sodium phosphate buffer. Cells were co-stained with anti-pericentrin using ab4448 at 1:500 dilution and ab7291 at 1:500 dilution. Alexa Fluor 633 goat anti mouse and Alexa Fluor 488 goat anti-rabbit (1:500 dilution) was used as secondary antibodies. DAPI was used as a nuclei counterstain. Representative images of mitotic cells with bipolar or multipolar spindles. ab7291 staining alpha Tubulin in SV40LT-SMC cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab7291 at a working concentration of 0.5 g/ml and ab190573, Rabbit monoclonal [EP1332Y] to alpha Tubulin(Alexa Fluor 647, shown in red) at 1/250 overnight at +4 C, followed by a further incubation at room temperature for 1h with an anti-mouse Alexa Fluor 488 (ab150117) at 2 g/ml (shown in green).Nuclear DNA was labelled in blue with DAPI.This product also gave a positive signal in 100% methanol (5 min) fixed SV40 cells under the same testing conditions.Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). IHC image of ab7291 staining alpha Tubulin in human colon formalin fixed paraffin embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab7291, 5ug/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre Overlay histogram showing HeLa cells stained with ab7291 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab7291, 1 g/1x106 cells) for 30 min at 22 C. The secondary antibody used was an anti-mouse DyLight 488 (ab96879) at 1/500 dilution for 30 min at 22 C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2 g/1x106 cells) used under the same conditions. Acquisition of 5,000 events was performed. ab7291 stainingalpha-Tubulin inCaco-2 cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab7291 at 1 g/ml and ab6046 at 1 g/ml overnight at +4 C, followed by a further incubation at room temperature for 1h with an anti-mouse Alexa Fluor 488 (ab150117) at 2 g/ml (shown in green) and anti-rabbit Alexa Fluor 594 (ab150088) at 2 g/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.Negative controls:1 Rabbit primary antibody and anti-mouse secondary antibody.2 Mouse primary antibody and anti-rabbit secondary antibody.Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used. ab7291 stainingalpha TubulininNIH3T3 cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab7291 at 1 l/ml and ab6046 at 1 g/ml overnight at +4 C, followed by a further incubation at room temperature for 1h with an anti-mouse Alexa Fluor 488 (ab150117) at 2 g/ml (shown in green) and anti-rabbit Alexa Fluor 594 (ab150088) at 2 g/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.Negative controls:1 Rabbit primary antibodyand anti-mouse secondary antibody.2 Mouse primary antibody and anti-rabbit secondary antibody.Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used. Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)This image is courtesy of an AbReview submitted by Elena Kashuba. All lanes : Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/2500 dilutionLanes 1-2 : Daudi (Human Burkitt\'s lymphoma cell line) at 10 µgLanes 3-4 : Daudi (Human Burkitt\'s lymphoma cell line) at 15 µgLanes 5-6 : Daudi (Human Burkitt\'s lymphoma cell line) at 20 µgSecondaryAll lanes : HRP conjugated monoclonal Goat Anti-Mouse IgG at 1/1000 dilutionPerformed under reducing conditions.Predicted band size: 50 kDaObserved band size: 50 kDaExposure time: 1 minuteSee Abreview All lanes : Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1 µg/mlLane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma cell line) whole cell lysateLane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysateLane 3 : PC12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysateLysates/proteins at 10 µg per lane.SecondaryAll lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/50000 dilutionDeveloped using the ECL technique.Performed under reducing conditions.Predicted band size: 50 kDaObserved band size: 50 kDaExposure time: 150 secondsThis blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab7291 overnight at 4 C. Antibody binding was detected using an anti-mouse HRP (ab97040), and visualised using ECL development solution ab133406 FABP4 (green) was detected using FABP4 primary antibody (ab92501; diluted 1/1000). Alpha tubulin (red) was detected usingthe mouse monoclonal (ab7291) antibody. Cells were imaged by confocal microscopy, using z-stack for adipocyte-like cells. IHC image of ab7291 staining alpha Tubulin in rat colon formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab7291, 0.5ug/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times. ICC/IF image of ab7291 stained human HeLa cells. The cells were methanol fixed (5 min)and incubated with the antibody (ab7291, 1 g/ml) for 1h at room temperature. The secondary antibody (green)was Alexa Fluor 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor 594 WGA was used to label plasma membranes (red). Immunofluorescent imaging of human cells (U2OS) with ab7291 reveals a delicate network of alpha-tubulin (green) located exclusively in the cytoplasm. The nucleus is stained blue.IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour. Primary antibody used at 1/200 in 5% milk / 0.2% TWEEN for one hour, secondary antibody Alexa 488 for 30 minutes. All blocking and incubation steps carried out at 37 degrees. Publishing research using ab7291? Please let us know so that we can cite the reference in this datasheet. ab7291 has been referenced in 758 publications. Dirks ML et al. Short-term bed rest-induced insulin resistance cannot be explained by increased mitochondrial H2 O2 emission. J Physiol 598:123-137 (2020). HumanPubMed: 31721213 Yan J et al. Integrative transcriptomic and proteomic analysis reveals CD9/ITGA4/PI3K-Akt axis mediates trabecular meshwork cell apoptosis in human glaucoma. J Cell Mol Med 24:814-829 (2020).PubMed: 31680442 Miao L et al. Targeting the STING pathway in tumor-associated macrophages regulates innate immune sensing of gastric cancer cells. Theranostics 10:498-515 (2020).PubMed: 31903134 Leylek TR et al. Reduced Expression of Genes Regulating Cohesion Induces Chromosome Instability that May Promote Cancer and Impact Patient Outcomes. Sci Rep 10:592 (2020).PubMed: 31953484 Dave KM et al. Characterization of the SIM-A9 cell line as a model of activated microglia in the context of neuropathic pain. PLoS One 15:e0231597 (2020).PubMed: 32287325 View all Publications for this product Blocking step BSA as blocking agent for 30 minute(s) Concentration: 5% Temperature: 25 C Blocking step BSA as blocking agent for 10 minute(s) Concentration: 1% Temperature: 25 C Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) Concentration: 5% Temperature: 20 C Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) Concentration: 5% Temperature: 22 C Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) Concentration: 5% Temperature: 25 C Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) Concentration: 5% Temperature: 25 C Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) Concentration: 5% Temperature: 25 C Blocking step Licor intercept TBS as blocking agent for 16 hour(s) and 0 minute(s) Concentration: 5% Temperature: 4 C Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) Concentration: 3% Temperature: RT C Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) Concentration: 1% Temperature: 25 CPlease note: All products are FOR RESEARCH USE ONLY. 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