Anti-βIII Tubulin antibody ValidAbTM
(HB8947)
Product overview
| Name | Anti-βIII Tubulin antibody ValidAbTM |
| Host | Chicken |
| Clonality | Polyclonal |
| Target | Beta III tubulin |
| Description | Antibody to βIII Tubulin - cytoskeletal protein used as a neuronal marker. Part of the ValidAb™ range of highly validated, data-rich antibodies. |
Write Your Own Review
Validation data
Figure 1. βIII tubulin staining with HB8947 of cultured rat hippocampal neurons.
βIII-tubulin staining using HB8947 reveals the complex network of neural processes in a cultured rat hippocampal neuron preparation. Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by being blocked in 1% BSA, 300mM glycine. HB8947 (chicken polyclonal anti-βIII-tubulin) was incubated overnight (4°C) at a 1:2,000 dilution (0.15µg/ml). This was followed by a one-hour incubation with a polyclonal goat anti-chicken Alexa Fluor 488 conjugated secondary antibody at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 40x objective (1.28x zoom), 405nm (1% power, PMT: 666V gain) and 488nm (1% power, Hyd: 14% gain) lasers in a z-stack (0.27µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. βIII tubulin staining with HB8947 of cultured rat hippocampal neurons.
βIII-tubulin staining using HB8947 reveals the complex network of neural processes in a cultured rat hippocampal neuron preparation. Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by being blocked in 1% BSA, 300mM glycine. HB8947 (chicken polyclonal anti-βIII-tubulin) was incubated overnight (4°C) at a 1:2,000 dilution (0.15µg/ml). This was followed by a one-hour incubation with a polyclonal goat anti-chicken Alexa Fluor 488 conjugated secondary antibody at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 40x objective (1.28x zoom), 405nm (1% power, PMT: 663V gain) and 488nm (2.25% power, Hyd: 16.5% gain) lasers in a z-stack (0.32µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Cultured neurons expressing both βIII tubulin and VLP1.
Colocalization of βIII-tubulin and the neuronal protein Visinin-like protein 1 (VLP1) in cultured rat hippocampal neurons. Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by being blocked in 1% BSA, 300mM glycine. HB8947 (chicken polyclonal anti-βIII-tubulin) and a mouse monoclonal anti-VLP1 antibody were incubated overnight (4°C) both at a 1:1,000 dilution. This was followed by a one-hour incubation with polyclonal goat anti-chicken Alexa Fluor 488 conjugated and polyclonal goat anti-mouse Dy550 conjugated secondary antibodies at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured as a tilescan using a Leica DMI6000 inverted epifluorescence microscope with a Photometrics Prime 95B camera. The image was captured using a 40x objective with DAP (5ms exposure), L5 (25ms exposure) and RHO (10ms exposure) filters. Following capture, the image was deconvolved using Huygens software before having the stack flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. Concentration response of HB8947 staining in cultured rat hippocampal neurons.
HB8947 effectively stains neurons for βIII-tubulin at dilutions as low as 1:8,000 with 1:2,000 being the recommended dilution. Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by being blocked in 1% BSA, 300mM glycine. HB8947 (chicken polyclonal anti-βIII-tubulin) was incubated overnight (4°C) at either a 1:1000 (300ng/ml), 1:2,000 (150ng/ml), 1:4,000 (75ng/ml) or 1:8,000 (32.5ng/ml) dilution. This was followed by a one-hour incubation with a polyclonal goat anti-chicken Alexa Fluor 488 conjugated secondary antibody at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a 40x objective as a tilescan using a Leica DMI6000 inverted epifluorescence microscope with a Photometrics Prime 95B camera. Exposure settings were as follows:
- 1:1,000 – 25ms
- 1:2,000 – 55ms
- 1:4,000 – 90ms
- 1:8,000 – 138ms
Figure 5. βIII tubulin staining with HB8947 of cultured rat hippocampal neurons.
βIII-tubulin staining using HB8947 reveals the complex network of neural processes in a cultured rat hippocampal neuron preparation. Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by being blocked in 1% BSA, 300mM glycine. HB8947 (chicken polyclonal anti-βIII-tubulin) was incubated overnight (4°C) at a 1:1,000 dilution (0.3µg/ml). This was followed by a one-hour incubation with a polyclonal goat anti-chicken Alexa Fluor 488 conjugated secondary antibody at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured as a tilescan using a Leica DMI6000 inverted epifluorescence microscope with a Photometrics Prime 95B camera. The image was captured using a 40x objective with DAP (5ms exposure), L5 (25ms exposure) and RHO (10ms exposure) filters. Following capture, the image was deconvolved using Huygens software before having the stack flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 6. Cultured neurons expressing both βIII tubulin and VLP1.
Colocalization of βIII-tubulin and the neuronal protein Visinin-like protein 1 (VLP1) in cultured rat hippocampal neurons. Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by being blocked in 1% BSA, 300mM glycine. HB8947 (chicken polyclonal anti-βIII-tubulin) and a mouse monoclonal anti-VLP1 antibody were incubated overnight (4°C) both at a 1:2,000 dilution. This was followed by a one-hour incubation with polyclonal goat anti-chicken Alexa Fluor 488 conjugated and polyclonal goat anti-mouse Dy550 conjugated secondary antibodies at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 40x objective (1.28x zoom), 405nm (1% power, PMT: 666V gain), 488nm (1% power, HyD: 14% gain) and 561nm (0.5% power, HyD: 10% gain) lasers in a z-stack (0.27µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 7. βIII tubulin staining with HB8947 of cultured rat hippocampal neurons.
βIII-tubulin staining using HB8947 reveals the complex network of neural processes in a cultured rat hippocampal neuron preparation. Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by being blocked in 1% BSA, 300mM glycine. HB8947 (chicken polyclonal anti-βIII-tubulin) was incubated overnight (4°C) at a 1:1,000 dilution (0.3µg/ml). This was followed by a one-hour incubation with a polyclonal goat anti-chicken Alexa Fluor 488 conjugated secondary antibody at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured as a tilescan using a Leica DMI6000 inverted epifluorescence microscope with a Photometrics Prime 95B camera. The image was captured using a 40x objective with DAP (5ms exposure), L5 (25ms exposure) and RHO (10ms exposure) filters. Following capture, the image was deconvolved using Huygens software before having the stack flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 1. βIII tubulin staining with HB8947 of cultured rat hippocampal neurons.
βIII-tubulin staining using HB8947 reveals the complex network of neural processes in a cultured rat hippocampal neuron preparation. Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by being blocked in 1% BSA, 300mM glycine. HB8947 (chicken polyclonal anti-βIII-tubulin) was incubated overnight (4°C) at a 1:2,000 dilution (0.15µg/ml). This was followed by a one-hour incubation with a polyclonal goat anti-chicken Alexa Fluor 488 conjugated secondary antibody at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 40x objective (1.28x zoom), 405nm (1% power, PMT: 666V gain) and 488nm (1% power, Hyd: 14% gain) lasers in a z-stack (0.27µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 2. βIII tubulin staining with HB8947 of cultured rat hippocampal neurons.
βIII-tubulin staining using HB8947 reveals the complex network of neural processes in a cultured rat hippocampal neuron preparation. Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by being blocked in 1% BSA, 300mM glycine. HB8947 (chicken polyclonal anti-βIII-tubulin) was incubated overnight (4°C) at a 1:2,000 dilution (0.15µg/ml). This was followed by a one-hour incubation with a polyclonal goat anti-chicken Alexa Fluor 488 conjugated secondary antibody at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 40x objective (1.28x zoom), 405nm (1% power, PMT: 663V gain) and 488nm (2.25% power, Hyd: 16.5% gain) lasers in a z-stack (0.32µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 3. Cultured neurons expressing both βIII tubulin and VLP1.
Colocalization of βIII-tubulin and the neuronal protein Visinin-like protein 1 (VLP1) in cultured rat hippocampal neurons. Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by being blocked in 1% BSA, 300mM glycine. HB8947 (chicken polyclonal anti-βIII-tubulin) and a mouse monoclonal anti-VLP1 antibody were incubated overnight (4°C) both at a 1:1,000 dilution. This was followed by a one-hour incubation with polyclonal goat anti-chicken Alexa Fluor 488 conjugated and polyclonal goat anti-mouse Dy550 conjugated secondary antibodies at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured as a tilescan using a Leica DMI6000 inverted epifluorescence microscope with a Photometrics Prime 95B camera. The image was captured using a 40x objective with DAP (5ms exposure), L5 (25ms exposure) and RHO (10ms exposure) filters. Following capture, the image was deconvolved using Huygens software before having the stack flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 4. Concentration response of HB8947 staining in cultured rat hippocampal neurons.
HB8947 effectively stains neurons for βIII-tubulin at dilutions as low as 1:8,000 with 1:2,000 being the recommended dilution. Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by being blocked in 1% BSA, 300mM glycine. HB8947 (chicken polyclonal anti-βIII-tubulin) was incubated overnight (4°C) at either a 1:1000 (300ng/ml), 1:2,000 (150ng/ml), 1:4,000 (75ng/ml) or 1:8,000 (32.5ng/ml) dilution. This was followed by a one-hour incubation with a polyclonal goat anti-chicken Alexa Fluor 488 conjugated secondary antibody at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a 40x objective as a tilescan using a Leica DMI6000 inverted epifluorescence microscope with a Photometrics Prime 95B camera. Exposure settings were as follows:
- 1:1,000 – 25ms
- 1:2,000 – 55ms
- 1:4,000 – 90ms
- 1:8,000 – 138ms
Figure 5. βIII tubulin staining with HB8947 of cultured rat hippocampal neurons.
βIII-tubulin staining using HB8947 reveals the complex network of neural processes in a cultured rat hippocampal neuron preparation. Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by being blocked in 1% BSA, 300mM glycine. HB8947 (chicken polyclonal anti-βIII-tubulin) was incubated overnight (4°C) at a 1:1,000 dilution (0.3µg/ml). This was followed by a one-hour incubation with a polyclonal goat anti-chicken Alexa Fluor 488 conjugated secondary antibody at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured as a tilescan using a Leica DMI6000 inverted epifluorescence microscope with a Photometrics Prime 95B camera. The image was captured using a 40x objective with DAP (5ms exposure), L5 (25ms exposure) and RHO (10ms exposure) filters. Following capture, the image was deconvolved using Huygens software before having the stack flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 6. Cultured neurons expressing both βIII tubulin and VLP1.
Colocalization of βIII-tubulin and the neuronal protein Visinin-like protein 1 (VLP1) in cultured rat hippocampal neurons. Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by being blocked in 1% BSA, 300mM glycine. HB8947 (chicken polyclonal anti-βIII-tubulin) and a mouse monoclonal anti-VLP1 antibody were incubated overnight (4°C) both at a 1:2,000 dilution. This was followed by a one-hour incubation with polyclonal goat anti-chicken Alexa Fluor 488 conjugated and polyclonal goat anti-mouse Dy550 conjugated secondary antibodies at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured in Lightning deconvolution mode using a 40x objective (1.28x zoom), 405nm (1% power, PMT: 666V gain), 488nm (1% power, HyD: 14% gain) and 561nm (0.5% power, HyD: 10% gain) lasers in a z-stack (0.27µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Figure 7. βIII tubulin staining with HB8947 of cultured rat hippocampal neurons.
βIII-tubulin staining using HB8947 reveals the complex network of neural processes in a cultured rat hippocampal neuron preparation. Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by being blocked in 1% BSA, 300mM glycine. HB8947 (chicken polyclonal anti-βIII-tubulin) was incubated overnight (4°C) at a 1:1,000 dilution (0.3µg/ml). This was followed by a one-hour incubation with a polyclonal goat anti-chicken Alexa Fluor 488 conjugated secondary antibody at a 1:300 dilution. DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured as a tilescan using a Leica DMI6000 inverted epifluorescence microscope with a Photometrics Prime 95B camera. The image was captured using a 40x objective with DAP (5ms exposure), L5 (25ms exposure) and RHO (10ms exposure) filters. Following capture, the image was deconvolved using Huygens software before having the stack flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).
Product information
| Immunogen | Combination of three synthetic peptides derived from human MAP2 |
| Isotype | IgY |
| Purification | Immunogen affinity chromatography |
| Concentration | 0.3mg/ml |
| Formulation | Lyophilised. When reconstituted contains PBS with 0.02% sodium azide and 1% recombinant BSA. |
| Predicted species reactivity | Mouse, Rat, Human |
| Tested species reactivity | Mouse, Rat |
Tested applications
| Applications | ICC |
| ICC optimal concentration | 1:2,000 as tested in cultured rat neurons |
| Positive control | ß3-tubulin is widely expressed in neural tissues. It is also well expressed in SH-SY5Y, Hep G2, A549 and SCLC-21H cell lines. |
| Negative control | Non-neural tissues, except for tissue from the testes. Poorly expressed in many cell lines such as JURKAT, HeLa and HEK293. |
| Open data link | Please follow this link to OSF |
Target information
| Other names | TUBB3, Tubulin beta-4 chain, Tubulin beta-III |
| UniProt ID | Q13509 |
| Structure image |  |
| Gene name | TUBB3 |
| NCBI full gene name | tubulin beta 3 |
| Entrez gene ID |
| Amino acids | 450 (50.4kDa) |
| Isoforms | Beta III tubulin has two isoforms. Isoform 1: canonical; Isoform 2: missing amino acids 1-72 |
| Expression | Beta III tubulin is expressed almost exclusively within neurones present in the central nervous system and peripheral nervous system. Expression has also been found within the sertoli cells of the testes. |
| Subcellular expression | Beta III tubulin is a key cytoskeletal component therefore is widely expressed as bundles of Beta III tubulin positive fibres. |
| Processing | Following translation no processing is required for Beta III tubulin to reach its active conformation. |
| Post translational modifications | Beta III tubulin is subject to three postranslational modifications: phosphorylation by CDK1 at Ser172, Polyglutamylation at Glu438 and phosphorylation at Ser 444 (note: this is within the epitope of HB6639) |
| Homology (compared to human) | Mouse and human proteins are identical while rat beta III tubulin shows a single change (E440D) |
| Similar proteins | Beta III tubulin shows similarity in a BLAST search to other beta tubulin family members (e.g. Tubulin beta IV 100%, tubulin beta VI 96%, tubulin beta IIA 95%, tubulin beta IIB 95%) alongside alpha tubulin (96% similarity) and epididymis sperm binding protein (95%) |
Storage & Handling
| Storage instructions | -20°C then use reconstitution advice |
| Reconstitution advice | We recommend reconstituting with either:
Take care when opening as the precipitate is extremely light and can easily be lost if disturbed. When reconstituting make sure that the antibody is thoroughly dissolved by pipetting up and down before giving the antibody a brief spin at <10,000g to make sure that all material is recovered and at the bottom of the tube. For more information please see our detailed guide on storing and using your antibody |
| Important | This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use |
Technical guides
Western Blot Protocol (1 MB) Immunocytochemistry Protocol (1.2 MB) Immunohistochemistry Protocol (1.6 MB) References for Anti-βIII Tubulin antibody ValidAbTM
References are publications that support the biological activity of the product
-
Class III beta-tubulin expression and in vitro resistance to microtubule targeting agents.
Stengel C et al (2010) British journal of cancer 102 : 316-24 -
Mutations in the neuronal ß-tubulin subunit TUBB3 result in malformation of cortical development and neuronal migration defects.
Poirier K et al (2010) Human molecular genetics 19 : 4462-73 -
Proteomic characterization of cytoskeletal and mitochondrial class III beta-tubulin.
Cicchillitti L et al (2008) Molecular cancer therapeutics 7 : 2070-9
Related Products
New Products in this Area
Related Products
- Code:
- HB6639
Antibody to βIII Tubulin - cytoskeletal protein used as a neuronal marker. Part of the ValidAb™ range of highly validated, data-rich antibodies.
- Code:
- HB6581
Antibody to MAP2 - cytoskeletal protein used as a neuronal marker. Part of the ValidAb™ range of highly validated, data-rich antibodies.
- Code:
- HB9587
Antibody to MAP2 - cytoskeletal protein used as a neuronal marker. Part of the ValidAb™ range of highly validated, data-rich antibodies.