Anti-β-tubulin antibody ValidAb™

(HB6491)
Technical documents: SDS Datasheet

Product overview

Name Anti-β-tubulin antibody ValidAb™
Host Mouse
Clonality Monoclonal
Target β-tubulin
Description

Antibody to β-tubulin - cytoskeletal component widely used for imaging microtubules and as a loading control. Part of the ValidAb™ range of highly validated, data-rich antibodies.

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Validation data

Figure 1. ß-Tubulin and Neurofilament L expression in cultured rat neurones

HB6491 stained the dense network of tubulin filaments present in neurones cultured from P2 rats. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6491 was incubated overnight (4°C) at a 1:2000 dilution (0.5µg/ml) alongside HB7266 (1:4,000 dilution, 0.25 µg/ml) followed by a one hour incubation with secondary antibodies (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution and polyclonal goat anti-rabbit DyLight 594 conjugated, Thermofisher 35511, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using Lightning adaptive deconvolution using a 63x objective and 405nm (11.8% power, PMT: 817V gain), 496nm (0.5% power, HyD: 10% gain) and 561nm (2.0% power, HyD: 17.6% gain) lasers. Images were captured as a stack (0.26µm z-spacing) being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 2. Concentration response of HB6491 staining in a rat brain cytosol preparation

HB6491 shows consistent results with low background at dilutions as low as 1:64,000 (15.6 ng/ml). Method: cytosol fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). Rat cytosol samples (20µg / lane) were loaded onto a 12% acrylamide gel alongside a protein ladder (NEB colour pre-stained broad range, P7719S) before being run at 60V for 40 minutes followed by 120V for 100 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. Following transfer the membrane was cut into strips using Ponceau dye to visualise and cut individual lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6491. Each strip was incubated separately with a separate HB6491 concentration with this ranging from 1µg/ml (1:1,1000 dilution) to 3.9ng/ml (1:256,000 dilution). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Band intensity was calculated using Image Studio version 5.2.5 (LiCor) and a graph was constructed in GraphPad Prism 9 using a 3-parameter Hill equation curve fit.

Figure 3. ß-Tubulin expression in various tissue lysates and preparations

HB6491 revealed a single band of size 56.5kDa present in all samples with a strong signal to noise ratio. Due to the higher expression in brain samples the membrane was imaged at two different exposure levels which are presented side by side. Method: mouse brain and rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation). Samples (20µg / lane) were loaded onto a 12% acrylamide gel alongside a protein ladder (NEB colour pre-stained broad range, P7719S) before being run at 60V for 54 minutes followed by 120V for 1hr 46min. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6491 at a 1:5000 dilution (0.2µg/ml). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Following imaging, the membrane was stripped by incubation with two changes of stripping buffer (HB7756) then was re-probed following the protocol described with HB9177 (mouse monoclonal anti-GAPDH) at a 1:4000 dilution (250ng/ml) overnight at 4°C. Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs and visualised again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 4 min exposure, 700nm channel: 30 sec exposure).

Figure 4. ß-Tubulin and Neurofilament L expression in cultured rat neurones.

HB6491 stained the dense network of tubulin filaments present in neurones cultured from P2 rats. Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6491 was incubated overnight (4°C) at a 1:2000 dilution (0.5µg/ml) alongside HB7266 (1:4,000 dilution, 0.25 µg/ml) followed by a one hour incubation with secondary antibodies (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution and polyclonal goat anti-rabbit DyLight 594 conjugated, Thermofisher 35511, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using Lightning adaptive deconvolution using a 63x objective and 405nm (11.8% power, PMT: 937V gain), 496nm (0.5% power, HyD: 11.8% gain) and 561nm (2.0% power, HyD: 22.9% gain) lasers. Images were captured as a stack (0.35µm z-spacing) being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 5. The effect of varying HB6491 concentration upon ß-tubulin staining in cultured rat neurones.

HB6491 produces consistent staining with a high signal to noise ratio at concentrations as low at 62.5ng/ml (1:16,000 dilution). Method: neurones were cultured from PND2 rats following established protocols (Brewer and Torricelli, 2007. Nat Protoc 2, 1490–1498) and fixed with 4% PFA on DIV21. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6491 was incubated overnight (4°C) at dilutions ranging from 1:2000 (500ng/ml) to 1:16,000 (125ng/ml). All incubations additionally included HB7266 (mouse monoclonal anti-neurofilament L) at a concentration of 250ng/ml (1:4,000 dilution). This was followed by a one hour incubation with secondary antibodies (Polyclonal goat anti-mouse DyLight 488 conjugated, Thermofisher 35503, 1:300 dilution and polyclonal goat anti-rabbit DyLight 594 conjugated, Thermofisher 35511, 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 in a z-stack using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope in Lightning adaptive deconvolution mode using a 63x objective. Laser powers and gains were as follows:

  • 1:2,000 – 405nm (11.8% power, PMT: 937V gain), 496nm (0.5% power, HyD: 11.8% gain) and 561nm (2.0% power, HyD: 22.9% gain)
  • 1:4,000 - 405nm (11.8% power, PMT: 825V gain), 496nm (0.5% power, HyD: 10.2% gain) and 561nm (2.0% power, HyD: 14.1% gain)
  • 1:8,000 - 405nm (11.8% power, PMT: 825V gain), 496nm (0.5% power, HyD: 10.2% gain) and 561nm (2.0% power, HyD: 14.1% gain)
  • 1:1,6000  - 405nm (11.8% power, PMT: 825V gain), 496nm (0.5% power, HyD: 12.7% gain) and 561nm (1.0% power, HyD: 10.0% gain)

Images were processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool followed by Z-projection, stacking and montage creation. 

Figure 6. ß-tubulin and phalloidin staining in HeLa cells.

HB6491 revealed the dense network of tubulin containing microtubules within HeLa cells while FITC phalloidin (HB0814) stains actin filaments. Methods: HeLa cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6491 was incubated overnight (4°C) at a 1:2000 dilution (0.5µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse Janelia Fluor 549 (HB7336) conjugated secondary antibody (1:300 dilution) and FITC Phalloidin (HB0814, 183nM). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using Lightning adaptive deconvolution using a 63x objective and 405nm (11.1% power, PMT: 500V gain), 488nm (2.4% power, HyD: 22.3% gain) and 561nm (3.0% power, HyD: 22.3% gain) lasers. Images were captured as a stack (0.37µm z-spacing) being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 7. ß-tubulin and phalloidin staining in HeLa cells.

HB6491 revealed the dense network of tubulin containing microtubules within HeLa cells while FITC phalloidin (HB0814) stains actin filaments. Methods: HeLa cells were cultured following standard protocols in 10% FBS in DMEM with 1% pen-Strep before being fixed in 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6491 was incubated overnight (4°C) at a 1:2000 dilution (0.5µg/ml) followed by a one hour incubation with a polyclonal goat anti-mouse Janelia Fluor 549 (HB7336) conjugated secondary antibody (1:300 dilution) and FITC Phalloidin (HB0814, 183nM). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. The image was captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. The image was captured using Lightning adaptive deconvolution using a 63x objective and 405nm (11.1% power, PMT: 500V gain), 488nm (2.4% power, HyD: 35.0% gain) and 561nm (3.0% power, HyD: 22.3% gain) lasers. Images were captured as a stack (0.46µm z-spacing) being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Product information

Immunogen

Tubulin preparation from pig brain

Clone number 1B12
Isotype IgG2b
Purification

Protein G affinity purified

Concentration 1 mg/ml
Formulation 1:1 ratio of PBS:Glycerol + 5mM sodium azide
Predicted species reactivity Mouse, Rat, Human
Tested species reactivity Mouse, Rat, Human

Tested applications

Applications ICC, WB
Western blot optimal concentration

0.1μg/ml (1:10,000) as tested in rat brain cytosol fraction

ICC optimal concentration

0.25μg/ml (1:4,000) as tested in cultured primary rat neurones

Positive control

β-tubulin is expressed ubiquitously across nearly all mammalian cell and tissue types. It is also widely expressed in common cell lines (e.g. HEK293, SH-SY5Y, HeLa)

Negative control

β-tubulin is a cytoskeletal enzyme, so complete subcellular fractionation should be sufficient to provide a negative control. Due to its high expression, care should be taken to ensure that fractionation is complete without any cytoskeletal contamination.

Open data link

Please follow this link to OSF

Target information

Other names

Tubulin beta chain, Tubulin beta-5 chain, TUBB

UniProt ID P07437
Structure image  Chemical Structure
Gene name TUBB
NCBI full gene name tubulin beta class I
Entrez gene ID

203068

Amino acids

444 (49.7kDa)

Isoforms

β-tubulin has no isoforms other than the main sequence.

Expression

Expressed widely across all cell and tissue types including common cell lines.

Subcellular expression

Expressed in the cytoskeleton as a microtubule component.

Processing

None

Post translational modifications

β-tubulin has phosphorylation sites on multiple residues alongside numerous gamma-glutamylation sites.

Homology (compared to human)

Mouse and rat β-tubulin have a 98.4% and 93.2% identity to human β-tubulin as measured in a BLAST search

Similar proteins

No proteins (other than β-tubulin family members) show significant homology in a BLAST search

Storage & Handling

Storage instructions

-20°C

Important This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use

FAQs

What experiments can I use this β-tubulin antibody as a loading control in?

β-tubulin is expressed ubiquitously in all tissues however may not be appropriate if other proteins in the experiment are of a similar molecular weight. Please see our guide on choosing a loading control for more information.

What guarantee do you have that my β-tubulin antibody will perform as expected?

We guarantee that your β-tubulin antibody will work for the applications and species we list on the datasheet. If the antibody fails to perform as expected then we are happy to offer a 100% refund guarantee. For more details please see our guarantee policy.

Will my β-tubulin antibody work against species that have not been listed on the datasheet?

A species not being listed doesn’t mean that theβ-tubulin antibody won’t work, just that we haven’t tested it. If you test one of our antibodies in a new species please let us know (positive or negative)!

What protocols are available for use with this β-tubulin antibody?

We have made a comprehensive collection of protocols that we have used in our experiments to validate this β-tubulin antibody.

What counterstains do you recommend for use in ICC and IHC with this β-tubulin antibody?

We recommend using either DAPI or Hoechst 33342 to label cell nuclei. In some experiments it is also helpful to label actin filaments in the cytoskeleton using a Phalloidin conjugate such as FITC Phalloidin or Rhodamine Phalloidin-TRITC.

Any other questions?

For any other questions about our antibody products please see our technical FAQs for antibodies

References for Anti-β-tubulin antibody ValidAb™

References are publications that support the biological activity of the product
  • Tubulin: Structure, Functions and Roles in Disease.

    Binarová P et al (2019) Cells 8 :
  • The structured core of human β tubulin confers isotype-specific polymerization properties.

    Pamula MC et al (2016) The Journal of cell biology 213 : 425-33
  • beta-tubulin is a more suitable internal control than beta-actin in western blot analysis of spinal cord tissues after traumatic injury.

    Liu NK et al (2006) Journal of neurotrauma 23 : 1794-801
  • Free intermingling of mammalian beta-tubulin isotypes among functionally distinct microtubules.

    Lewis SA et al (1987) Cell 49 : 539-48