Recombinant Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (ab221935)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EP1569Y] to beta III Tubulin - BSA and Azide free
- Suitable for: IHC-P, ICC/IF, WB, Flow Cyt (Intra), mIHC
- Knockout validated
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free
See all beta III Tubulin primary antibodies -
Description
Rabbit monoclonal [EP1569Y] to beta III Tubulin - BSA and Azide free -
Host species
Rabbit -
Tested applications
Suitable for: IHC-P, ICC/IF, WB, Flow Cyt (Intra), mIHCmore details -
Species reactivity
Reacts with: Mouse, Rat, Human
Predicted to work with: Zebrafish -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: HCT116, PC12, HAP1, HEK293, and HeLa cell lysates and mouse brain, rat brain, mouse spinal cord, and rat spinal cord tissue lysates. IHC-P: Human breast carcinoma tissue. ICC/IF: NGF-differentiated PC12 cells. Flow Cyt (intra): U-87MG and HeLa cells. mIHC: Human cerebellum tissue
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General notes
ab221935 is the carrier-free version of ab52623.
Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.
This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.
Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.
This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.
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
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C. Do Not Freeze. -
Storage buffer
pH: 7.20
Constituent: PBS -
Carrier free
Yes -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EP1569Y -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
- HRP Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab190574)
- Alexa Fluor® 647 Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab190575)
- Biotin Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab195903)
- Alexa Fluor® 594 Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab201740)
- Alexa Fluor® 555 Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab202519)
- APC Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab224977)
- FITC Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab224978)
- PE Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab224979)
- Alexa Fluor® 488 Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab283728)
- Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)
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Compatible Secondaries
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Conjugation kits
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Isotype control
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KO cell lines
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KO cell lysates
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab221935 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 |
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IHC-P |
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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ICC/IF |
Use at an assay dependent concentration.
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WB |
Use at an assay dependent concentration. Detects a band of approximately 55 kDa.
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Flow Cyt (Intra) |
Use at an assay dependent concentration.
ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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mIHC |
Use at an assay dependent concentration.
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Notes |
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IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
ICC/IF
Use at an assay dependent concentration. |
WB
Use at an assay dependent concentration. Detects a band of approximately 55 kDa. |
Flow Cyt (Intra)
Use at an assay dependent concentration. ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
mIHC
Use at an assay dependent concentration. |
Target
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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
modificationsSome 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
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Database links
- Entrez Gene: 10381 Human
- Entrez Gene: 22152 Mouse
- Entrez Gene: 246118 Rat
- Omim: 602661 Human
- SwissProt: Q13509 Human
- SwissProt: Q9ERD7 Mouse
- SwissProt: Q4QRB4 Rat
- Unigene: 511743 Human
see all -
Alternative names
- beta 3 tubulin antibody
- beta 4 antibody
- beta-4 antibody
see all
Images
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All lanes : Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623) at 1/1000 dilution
Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : TUBB3 knockout HCT116 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 52 kDa why is the actual band size different from the predicted?This data was developed using the same antibody clone in a different buffer formulation (ab52623).
Lanes 1- 4: Merged signal (red and green). Green - ab52623 observed at 52 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab52623 was shown to react with beta III Tubulin in wild-type HCT116 cells in western blot. Loss of signal was observed when knockout cell line ab266900 (knockout cell lysate ab257070) was used. Wild-type HCT116 and TUBB3 knockout HCT116 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab52623 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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.
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Multiplex immunohistochemistry - Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (ab221935)
This data was developed using the same antibody clone in a different buffer formulation (ab52623).
Fluorescence multiplex immunohistochemical analysis of human cerebellum tissue (formalin-fixed paraffin-embedded section).
Merged staining of Neu-N (ab177487; yellow; Opal™570), anti-beta III Tubulin (ab52623; red; Opal™690) and anti-GFAP (ab68428; green; Opal™520).
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ kit.
The section was incubated in three rounds of staining with ab177487 (1/1000 dilution), ab52623 (1/200 dilution) and ab68428 (1/250 dilution); each using a separate fluorescent tyramide signal amplification system.
Sodium citrate antigen retrieval (pH 6.0) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.
DAPI (blue) was used as a nuclear counter stain.
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Immunocytochemistry/ Immunofluorescence - Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (ab221935)
This ICC/IF data was generated using the same anti-beta III tubulin antibody clone, EP1569Y, in a different buffer formulation (cat# ab52623).
ab52623 staining beta III Tubulin in wild-type HAP1 cells (top panel) and TUBB3 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab52623 at 1/500 dilution and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
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All lanes : Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : TUBB3 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 50 kDa why is the actual band size different from the predicted?This data was developed using the same antibody clone in a different buffer formulation (ab52623).
Lanes 1- 2: Merged signal (red and green). Green - ab52623 observed at 50 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab52623 was shown to react with beta III tubulin in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255358 (knockout cell lysate ab263857) was used. Wild-type HeLa and TUBB3 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab52623 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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.
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All lanes : Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623) at 1/1000 dilution
Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : Beta III Tubulin knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : HEK293 whole cell lysate
Lysates/proteins at 20 µg per lane.This WB data was generated using the same anti-beta III tubulin antibody clone, EP1569Y, in a different buffer formulation (cat# ab52623).
Lanes 1 - 4: Merged signal (red and green). Green - ab52623 observed at 52 kDa. Red - loading control, ab8245, observed at 37 kDa.
Ab52623 was shown to specifically react with beta III Tubulin in wild-type cells as signal was lost in beta III Tubulin knockout HAP1 cells. Wild-type and beta III Tubulin knockout samples were subjected to SDS-PAGE. Ab52623 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.
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Immunocytochemistry/ Immunofluorescence - Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (ab221935)
ab52623 staining beta III Tubulin in NGF-differentiated PC12 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 ab52623 at 5μg/ml and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Rabbit secondary (ab150081) at 2 μg/ml (shown in green) and AlexaFluor®594 Goat anti-Mouse secondary (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.
Negative controls: 1– Rabbit primary 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.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52623).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (ab221935)
ab52623, at a 1/50 dilution, staining human class III beta Tubulin in breast carcinoma tissue using Immunohistochemistry, Paraffin embedded tissue.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52623).
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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Flow Cytometry (Intracellular) - Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (ab221935)
Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labelling beta III Tubulin with ab52623 at 1/20 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. An Alexa Fluor®488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52623).
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Flow Cytometry (Intracellular) - Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (ab221935)
Overlay histogram showing U-87MG cells stained with ab52623 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 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 (ab52623, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (ab172730, 0.1µg/1x106) used under the same conditions. Unlabelled sample (blue line) was also used as a control.
Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
This antibody gave a positive signal in U-87MG cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52623).
Protocols
Datasheets and documents
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Datasheet download
Certificate of Compliance
References (6)
ab221935 has been referenced in 6 publications.
- Du J et al. Overexpression of Class III ß-tubulin, Sox2, and nuclear Survivin is predictive of taxane resistance in patients with stage III ovarian epithelial cancer. BMC Cancer 15:536 (2015). IHC ; Human . PubMed: 26198101
- da Silva TF et al. Peripheral nervous system plasmalogens regulate Schwann cell differentiation and myelination. J Clin Invest 124:2560-70 (2014). Mouse . PubMed: 24762439
- Yokomise H et al. Biomarkers as prognostic factors for cN2 or 3 non-small cell lung cancer treated by induction chemoradiotherapy and surgery. Anticancer Res 33:1107-15 (2013). Human . PubMed: 23482788
- Patel N et al. The growth factor receptor ERBB2 regulates mitochondrial activity on a signaling time scale. J Biol Chem 288:35253-65 (2013). WB . PubMed: 24142693
- Yokomise H et al. Chemotherapy followed by surgery on the basis of biomarker examination for patients with advanced non-small cell lung cancer. Anticancer Res 33:5597-602 (2013). PubMed: 24324104
- Lishko PV et al. Acid extrusion from human spermatozoa is mediated by flagellar voltage-gated proton channel. Cell 140:327-37 (2010). ICC/IF ; Human . PubMed: 20144758