Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-beta III Tubulin antibody [EPR19591] (ab215037)

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Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR19591] to beta III Tubulin
  • Suitable for: WB, ICC/IF, Flow Cyt, IP, IHC-P
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Overview

  • Product name

    Anti-beta III Tubulin antibody [EPR19591]
    See all beta III Tubulin primary antibodies

  • Description

    Rabbit monoclonal [EPR19591] to beta III Tubulin

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, ICC/IF, Flow Cyt, IP, IHC-Pmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide within Human beta III Tubulin aa 400 to the C-terminus. The exact sequence is proprietary.
    Database link: Q13509

  • Positive control

    • WB: Human cerebellum, breast cancer, fetal kidney and fetal brain lysates; U-87 MG, SH-SY5Y, HEK-293, C6, PC-12, Hap1 and HeLa whole cell lysates; Mouse brain lysate; Rat brain and heart lysates. IHC-P: Human cerebral cortex, glioma, cholangiocarcinoma and lung adenocarcinoma tissues; Mouse and rat cerebral cortex tissues. ICC/IF: SH-SY5Y and U-87 MG cells. Flow Cyt: U-87 MG cells. IP: U-87 MG whole cell lysate.

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

  • Form

    Liquid

  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.

  • Storage buffer

    pH: 7.2
    Preservative: 0.01% Sodium azide
    Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...

  • Purity

    Protein A purified

  • Clonality

    Monoclonal

  • Clone number

    EPR19591

  • Isotype

    IgG

  • Research areas

Applications

Our Abpromise guarantee covers the use of ab215037 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/2000. Detects a band of approximately 50 kDa (predicted molecular weight: 50 kDa).Can be blocked with Recombinant Human beta III Tubulin protein (ab140581).
ICC/IF 1/500.
Flow Cyt 1/200.
IP 1/30.
IHC-P 1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Target

  • 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. TUBB3 plays a critical role in proper axon guidance and mantainance.

  • Tissue specificity

    Expression is primarily restricted to central and peripheral nervous system.

  • Involvement in disease

    Defects in TUBB3 are the cause of congenital fibrosis of extraocular muscles type 3A (CFEOM3A) [MIM:600638]. A congenital ocular motility disorder marked by restrictive ophthalmoplegia affecting extraocular muscles innervated by the oculomotor and/or trochlear nerves. It is clinically characterized by anchoring of the eyes in downward gaze, ptosis, and backward tilt of the head. Congenital fibrosis of extraocular muscles type 3 presents as a non-progressive, autosomal dominant disorder with variable expression. Patients may be bilaterally or unilaterally affected, and their oculo-motility defects range from complete ophthalmoplegia (with the eyes fixed in a hypo- and exotropic position), to mild asymptomatic restrictions of ocular movement. Ptosis, refractive error, amblyopia, and compensatory head positions are associated with the more severe forms of the disorder. In some cases the ocular phenotype is accompanied by additional features including developmental delay, corpus callosum agenesis, basal ganglia dysmorphism, facial weakness, polyneuropathy.

  • Sequence similarities

    Belongs to the tubulin family.

  • Domain

    The highly acidic C-terminal region may bind cations such as calcium.

  • Post-translational
    modifications

    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.

  • Cellular localization

    Cytoplasm > cytoskeleton.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • beta 3 tubulin antibody
    • beta 4 antibody
    • beta-4 antibody
    • CDCBM antibody
    • CDCBM1 antibody
    • CFEOM3 antibody
    • CFEOM3A antibody
    • FEOM3 antibody
    • M(beta)3 antibody
    • M(beta)6 antibody
    • MC1R antibody
    • Neuron specific beta III Tubulin antibody
    • Neuron-specific class III beta-tubulin antibody
    • QccE-11995 antibody
    • QccE-15186 antibody
    • TBB3_HUMAN antibody
    • Tubb 3 antibody
    • TUBB3 antibody
    • TUBB4 antibody
    • Tubulin beta 3 antibody
    • Tubulin beta 3 chain antibody
    • Tubulin beta 4 antibody
    • Tubulin beta III antibody
    • Tubulin beta-3 chain antibody
    • Tubulin beta-4 chain antibody
    • Tubulin beta-III antibody
    • tuj 1 antibody
    • tuj1 antibody
    see all

Images

  • Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    All lanes : Anti-beta III Tubulin antibody [EPR19591] (ab215037) at 1/2000 dilution

    Lane 1 : Wild-type Hap1 cell lysate
    Lane 2 : TUBB3 knockout Hap1 cell lysate
    Lane 3 : Wild-type HeLa cell lysate
    Lane 4 : TUBB3 knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 50 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab215037 observed at 52 kDa. Red - loading control, ab8245 observed at 37 kDa.  

     ab215037 was shown to react with beta III Tubulin in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255358 (knockout cell lysate ab263857) was used. Wild-type and beta III Tubulin knockout samples were subjected to SDS-PAGE. ab215037 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 2000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

     

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

    Immunohistochemical analysis of paraffin-embedded human cholangiocarcinoma tissue labeling beta III Tubulin with ab215037 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution. 

    Cytoplasmic staining on human cholangiocarcinoma is observed.

    Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    All lanes : Anti-beta III Tubulin antibody [EPR19591] (ab215037) at 1/2000 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : TUBB3 knockout HAP1 whole cell lysate
    Lane 3 : HeLa whole cell lysate
    Lane 4 : HEK293 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 50 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab215037 observed at 50 kDa. Red - loading control, ab9484, observed at 37 kDa.

    ab215037 was shown to specifically react with TUBB3 in wild-type HAP1 cells as signal was lost in TUBB3 knockout cells. Wild-type and TUBB3 knockout samples were subjected to SDS-PAGE. ab215037 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1/2000 dilution and 1/20,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

  • Immunocytochemistry/ Immunofluorescence - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Immunocytochemistry/ Immunofluorescence - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) cells labeling beta III Tubulin with ab215037 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

    Confocal image showing cytoplasmic staining on U-87 MG cell line.

    The nuclear counterstain is DAPI (blue).

    Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)) at 1/200 dilution (red).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.

  • Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Lanes 1-5 : Anti-beta III Tubulin antibody [EPR19591] (ab215037) at 1/2000 dilution
    Lanes 6-7 : Anti-beta III Tubulin antibody [EPR19591] (ab215037) at 1/10000 dilution

    Lane 1 : Human cerebellum lysate at 20 µg
    Lane 2 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate at 20 µg
    Lane 3 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate at 20 µg
    Lane 4 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg
    Lane 5 : Human breast cancer lysate at 20 µg
    Lane 6 : Human fetal kidney lysate at 10 µg
    Lane 7 : Human fetal brain lysate at 10 µg

    Secondary
    All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

    Predicted band size: 50 kDa
    Observed band size: 50 kDa



    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: Lane 1/7: 1 second; Lane 2/3: 5 seconds; Lane 4/5: 3 minutes; Lane 6:30 seconds.

  • Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Lanes 1-2 : Anti-beta III Tubulin antibody [EPR19591] (ab215037) at 1/2000 dilution
    Lane 3 : Anti-beta III Tubulin antibody [EPR19591] (ab215037) at 1/5000 dilution
    Lanes 4-5 : Anti-beta III Tubulin antibody [EPR19591] (ab215037) at 1/10000 dilution

    Lane 1 : Mouse brain lysate
    Lane 2 : Rat brain lysate
    Lane 3 : Rat heart lysate
    Lane 4 : C6 (Rat glial tumor cell line) whole cell lysate
    Lane 5 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 50 kDa
    Observed band size: 50 kDa



    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: Lane 1/2: 1 second; Lane 3: 3 minutes; Lane 4/5: 30 seconds.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

    Immunohistochemical analysis of paraffin-embedded human cerebral cortex tissue labeling beta III Tubulin with ab215037 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution. 

    Cytoplasmic staining on human cerebral cortex is observed.

    Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

    Immunohistochemical analysis of paraffin-embedded human glioma tissue labeling beta III Tubulin with ab215037 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution. 

    Cytoplasmic staining on human glioma is observed.

    Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

    Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma tissue labeling beta III Tubulin with ab215037 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution. 

    Negative staining on human hepatocellular carcinoma. (PMID: 25039376).

    Counter stained with Hematoxylin.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

    Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma tissue labeling beta III Tubulin with ab215037 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution. 

    Cytoplasmic staining on human lung adenocarcinoma is observed.

    Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

    Immunohistochemical analysis of paraffin-embedded mouse cerebral cortex tissue labeling beta III Tubulin with ab215037 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution. 

    Cytoplasmic staining on mouse cerebral cortex is observed.

    Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP)  at 1/500 dilution.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

    Immunohistochemical analysis of paraffin-embedded rat cerebral cortex tissue labeling beta III Tubulin with ab215037 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.

    Cytoplasmic staining on rat cerebral cortex is observed.

    Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP)  at 1/500 dilution.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunocytochemistry/ Immunofluorescence - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Immunocytochemistry/ Immunofluorescence - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SH-SY5Y (Human neuroblastoma cell line from bone marrow) cells labeling beta III Tubulin with ab215037 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

    Confocal image showing cytoplasmic staining on SH-SY5Y cell line.

    The nuclear counterstain is DAPI (blue).

    Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)) at 1/200 dilution (red).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.

  • Flow Cytometry - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Flow Cytometry - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

    Flow cytometric analysis of 4% paraformaldehyde-fixed U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) cells labeling beta III Tubulin with ab215037 at 1/200 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor® 488) at 1/2000 dilution was used as the secondary antibody.

  • Immunoprecipitation - Anti-beta III Tubulin antibody [EPR19591] (ab215037)
    Immunoprecipitation - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

    beta III Tubulin was immunoprecipitated from 0.35 mg of U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate with ab215037 at 1/30 dilution.

    Western blot was performed from the immunoprecipitate using ab215037 at 1/1000 dilution.

    VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution

    Lane 1: U-87 MG whole cell lysate 10µg (Input).

    Lane 2: ab215037 IP in U-87 MG whole cell lysate.

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab215037 in U-87 MG whole cell lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    Exposure time: 10 seconds.

References (0)

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