Overview

  • Product name
    Anti-beta III Tubulin antibody [TU-20]
    See all beta III Tubulin primary antibodies
  • Description
    Mouse monoclonal [TU-20] to beta III Tubulin
  • Host species
    Mouse
  • Specificity

    Class III beta-tubulin specific for neurons.

  • Tested applications
    Suitable for: ICC, IHC-P, WB, IHC-FoFr, IHC-Fr, ICC/IF, IHC-P, IP, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Hamster, Cow, Cat, Human, Pig, Carp, Baboon
    Predicted to work with: a wide range of other species, Mammals
  • Immunogen

    Synthetic peptide corresponding to Human beta III Tubulin aa 441-448 conjugated to Keyhole Limpet Haemocyanin (KLH).
    Sequence:

    ESESQGPK

  • Epitope
    ESESQGPK (aa 441-448)
  • Positive control
    • ICC/IF: Neuro-2a cells. WB: Human brain and spinal cord tissue lysate. IHC-P: Human cerebellum tissue. Flow Cyt: SH-SY5Y cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab7751 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
ICC 1/500.
IHC-P Use a concentration of 10 µg/ml. Pretreatment: 0.1% pepsin (trypsin) in 0.1 M HCl for 30 minutes at RT. High-temperature citrate buffer antigen retrieval can also be performed.
WB Use a concentration of 1 - 2 µg/ml. We suggest reducing conditions and a 90 minute incubation time.
IHC-FoFr Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.
ICC/IF 1/1000.
IHC-P Use a concentration of 5 µg/ml.
IP Use at an assay dependent concentration.
Flow Cyt 1/20 - 1/50.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

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
  • Alternative names
    • beta 3 tubulin 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
    see all

Images

  • Naive hOMSC express constitutively neuronal and dopaminergic markers.

    Immunofluorescence of neuronal (A–C) and dopaminergic (D–F) markers in naïve hOMSC, scale bars 100 µM.

    Cells obtained from two donors (24 and 35 years old) were fixed in 4% PFA-PBS and pre-incubated for 60 min in blocking solution (5% goat serum, 1% BSA, 0.05% Triton-X in PBS). Primary antibodies were diluted in the blocking solution and applied overnight at 4°C. The following primary antibodies were used, β-III-Tubulin (1∶200, ab7751, abcam), Map2 (1∶200), synapsin (1∶200), tyrosine hydroxylase (1∶200), Lmx1A (1∶200), Pitx3 (1∶200) and Nurr1 (1∶200).

    Primary antibodies were detected with fluorescent-labeled secondary antibodies Alexa®488 and 568 (1∶500,) for 1 h at room temperature. Nuclear DNA was stained using 4,6-diamino-2-phenylindole (DAPI) (1∶1000).

  • Kif2a is involved in the differentiation of NSCs/NPCs.

    (Panel A) Mouse embryos were electroporated with indicated plasmids (scramble shRNA, Kif2a shRNA1 or Kif2a shRNA1+Kif2aRes) at E13.5, and analyzed at E15.5. GFP (green) represents cells expressing the indicated plasmids; TU20 (ab7751, red) represents immature neurons. Scale bars = 50 μm.

  • Confirmation of differentiation of iPSCs into cortical neuronal cultures.

    Images taken from clones C2 (trisomic) and C3 -D21 (disomic) 40 days after initiation of the differentiation protocol. Fixed cells on chamber slides were probed with antibodies against the marker proteins listed in the left column. Neuronal marker Beta III tubulin is red (ab7751); DAPI is blue; and Ctip2, TBR1, SLC17A7 (vGLUT1) and GRIK2 are green. Size bar = 20 μ.

  • ab7751 staining beta III Tubulin in Neuro-2a (Mouse neuroblastoma cell line) 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 ab7751 at 1/1000 and ab6046 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Mouse secondary (ab150117) at 2 μg/ml (shown in green) and AlexaFluor®594 Goat anti-Rabbit secondary (ab150088) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labeled 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.

  • All lanes : Anti-beta III Tubulin antibody [TU-20] (ab7751) at 1 µg/ml

    Lane 1 : Human brain tissue lysate - total protein (ab29466) at 20 µg
    Lane 2 : Human spinal cord tissue lysate - total protein (ab29188)

    Secondary
    All lanes : IRDye® 800CW Goat Anti-Mouse at 1/10000 dilution

    Performed under reducing conditions.

    Observed band size: 51 kDa
    why is the actual band size different from the predicted?



    This 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 LI-COR® blocking buffer before being incubated with ab7751 overnight at 4°C. Antibody binding was detected using the IRDye® 800CW Goat Anti-Mouse secondary at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Odyssey® CLx Imaging System.

  • IHC image of ab7751 staining beta III Tubulin in human cerebellum 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 (pH 6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab7751, 5μg/ml, 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 hematoxylin 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.

  • ab7751 staining beta III Tubulin in induced neurons derived from human pluripotent cells by ICC/IF (Immunocytochemistry/immunofluorescence).

    Cells were fixed with paraformaldehyde, permeabilized with 0.25% Triton X-100 in PBS and blocked with 1% BSA for 30 minutes at 22°C. Samples were incubated with primary antibody (1/500) for 16 hours at 4°C. An Alexa Fluor® 594-conjugated Donkey anti-mouse IgG polyclonal (1/1000) was used as the secondary antibody.

    See Abreview

  • Overlay histogram showing SH-SY5Y (Human neuroblastoma cell line from bone marrow) cells stained with ab7751 (red line).

    The cells were fixed with 4% paraformaldehyde (10 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 (ab7751, 1/50 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

    This antibody gave a positive signal in SH-SY5Y cells fixed with 80% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

  • ab7751 staining neuron specific beta III Tubulin in human colon tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).

    Tissue underwent formaldehyde fixation before heat mediated antigen retrieval in citric acid pH 6 and then blocking with 1% BSA was performed for 10 minutes at RT. The primary antibody was used at dilution at 1/200 and incubated with sample in TBS/BSA/azide for 2 hours. A biotin conjugated goat polyclonal to mouse IgG was used at dilution at 1/200 as secondary antibody. Images A and B represent staining of the clone TU20 and 2G10 respectively in ganglia of Auerbach's plexus.

    See Abreview

  • Immunohistochemistical detection of neuron specific beta III Tubulin using antibody (ab7751) on whole mouse e12 embryo (formaldehyde fixed/frozen sections).

    Permeabilization: No. Blocking step: 1% BSA for 10 mins @ rt°C. Primary Antibody Dilution 1/200; Incubation time 2 hours in TBS/BSA/azide. Secondary antibody: anti Mouse IgG Conjugated to Alexa Fluor® 594 (1/1000).

    Floorplate region of developing cervical spinal cord (I have not studied this particular region).

    See Abreview

  • ab7751 immunostaining (1/500) neuronal processes in primary mouse cortical culture.

  • Immunohistochemical detection with neuron specific beta III Tubulin antibody [TU-20] (ab7751) on formaldehyde fixed rat spinal cord tissue sections.

    Antigen retrieval step: heat mediated in citric acid pH 6. Blocking: 1% BSA for 10 mins@ rt°C. Primary antibody ab7751 incubated at 1/500 for 2 hours. Secondary antibody: anti mouse IgGs conjugated to biotin (1/200).

    ab7751 shows good fibrillar positivity in rat spinal motor neurone cell bodies. Some L/S nerve fibre processes are clearly seen yet the majority in the white matter are also not obvious.

    See Abreview

References

This product has been referenced in:
  • Han S  et al. Electrical stimulation accelerates neurite regeneration in axotomized dorsal root ganglion neurons by increasing MMP-2 expression. Biochem Biophys Res Commun 508:348-353 (2019). Read more (PubMed: 30503336) »
  • Xu D  et al. MEKK3 coordinates with FBW7 to regulate WDR62 stability and neurogenesis. PLoS Biol 16:e2006613 (2018). Read more (PubMed: 30566428) »
See all 92 Publications for this product

Customer reviews and Q&As

1-10 of 15 Q&A

Answer

Thank you for contacting us.

I think either of the two beta III tubulin clones will be effective, but I would choose ab7751 (the TU-20 clone) simply because it has been published in more references than the other clone, ab78078 (2G10 clone).

For staining neurofilaments, I recommend ab72997, anti-68kDa Neurofilament.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information, for instance about which secondary antibodies you will need.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Answer

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for one vial of ab98787.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Question
Answer

DISCOUNT CODE: XXXXXXXXXX
Expiration date: 17-09-2012

I am very pleased to hear you would like to use ab7751 inWB and send us an image of your results.

This code will give you 1 free [PRIMARY ANTIBODY/PROTEIN] before the expiration date. To redeem this offer, please submit an Abreview with the image forWB and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

The code will be active once the Abreview with the image has been submitted and can be redeemed in one of the following ways:
1) Call to place your order and mention the code to our customer service department;
2) Include the code in your fax order;
3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated. Remember if the product does not work you will be covered by our Abpromise guarantee and eligible for a full refund or replacement.

If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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Answer

Vielen Dank für Ihre Anrufe.

Wie versprochen habe ich alle unsere Langerin- bzw. TACD2-Antikörper danach geprüft, ob sie schon einmal mit Schwein getestet wurden - was leider bei keinem der Fall ist.

Daher habe ich die Immunogene auf Sequenzähnlichkeit mit dem Langerin bzw. TACD2 des Schweins (SwissProt IDs B3FVQ1 bzw. F1S7A4) untersucht, und die BLAST-Recherchen ergaben folgendes:

Langerin

ab68434 (Immunogen Aminosäuren 1-20, MTVEKEAPDAHFTVDKQNIS): 83%

https://www.abcam.com/index.html?datasheet=68434 (or use the following: https://www.abcam.com/index.html?datasheet=68434).

ab125476: 79%

https://www.abcam.com/index.html?datasheet=125476 (or use the following: https://www.abcam.com/index.html?datasheet=125476).

ab90959: 66%

https://www.abcam.com/index.html?datasheet=90959 (or use the following: https://www.abcam.com/index.html?datasheet=90959).

ab22111: viele Treffer mit unterschiedlichen Ähnlichkeiten (ca. 70%), daher nicht empfehlenswert zu testen

https://www.abcam.com/index.html?datasheet=22111 (or use the following: https://www.abcam.com/index.html?datasheet=22111).

TACD2

ab65006 (Aminosäuren 130-180): 92%

https://www.abcam.com/index.html?datasheet=65006 (or use the following: https://www.abcam.com/index.html?datasheet=65006).

ab58930 (Immunogen Aminosäuren 194-209, VHYEQPTIQIELRQNT): 86%

https://www.abcam.com/index.html?datasheet=58930 (or use the following: https://www.abcam.com/index.html?datasheet=58930).

ab77551: 85%

https://www.abcam.com/index.html?datasheet=77551 (or use the following: https://www.abcam.com/index.html?datasheet=77551).

ab89928: 85%

https://www.abcam.com/index.html?datasheet=89928 (or use the following: https://www.abcam.com/index.html?datasheet=89928).

ab64320: Die Immunogensequenz muss im Labor nachgefragt werden.

https://www.abcam.com/index.html?datasheet=64320 (or use the following: https://www.abcam.com/index.html?datasheet=64320).

Für diese Antikörper kann ich Ihnen wie zuvor wieder jeweils einen Testrabatt anbieten, d.h. wenn Sieeinen der Antikörper kaufen, uns Ihre Ergebnisse mit Schwein per Abreview mitteilen, bekommen Sie wieder jeweils einen kosten Primärantikörper zum Dank (und die Abpoints nicht zu vergessen) - https://www.abcam.com/collaborationdiscount.

Dies wäre vor allem für den TACD2- Antikörper ab65006 zu empfehlen, den wir schon in der Immunhistochemiegetestet haben und daher wissen, dass er wenigstensin der Anwendung funktioniert - auch wenn die Spezies Schwein noch nichtvalidiert wurde. Allerdings ist mit einer Sequenzähnlichkeit von 92%die Wahrscheinlichkeit einer Kreuzreaktivität sehr hoch.Aus dem gleichen Grund würde sich auch ab64320 eignen; hier können wir aber gerne noch die Rückmeldung aus dem Labor abwarten. Alle anderen Antikörper wurden bisher noch nicht in der Immunhistochemie getestet, daher besteht hier ein doppeltes Risiko, da wir nicht wissen, ob die Antikörper für diese Anwendung geeignet sind.

Sie können diese Antikörper auch wie besprochen kostenlos erhalten, wenn Sie sie mittels der Gutscheine von den vorherigen Testungen bestellen. Allerdings können wir in diesem Fall keine Garantie dafür übernehmen, wenn sienicht mit Schwein kreuzreagieren.

Ich hoffe, diese Informationen helfen Ihnen weiter. Falls Sie einen Rabattcode erhalten möchten oder weitere Fragen haben, zögern Sie bitte nicht, sich wieder an mich zu wenden.

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Answer

Thank you for your reply with this information.


Regarding your protocol - one thing that really stood out is the use of Triton as a permeabilizing agent. This detergent will also partially dissolve the nuclear membrane and will disrupt proteins when used at higher concentrations for longer periods of time. We typically recommend using it at 0.1-0.2% for 10 min only. Since both of these proteins are cytomplasmic, permeabilization with tween 20 is sufficient (I recommend 0.2 % for 10 - 15 minutes). This should decrease the amount of background and non-specific stain you are observing.


Additionally, I would recommend optimizing the conditions with each antibody individually rather than in sequential staining.


I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions or concerns.

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Answer

Thank you for contacting Abcam.


I am sorry that you have been experiencing difficulties with these antibodies in ICC. I have reviewed the images provided, but I was wondering if you would also provide a detailed summary of your protocol and sample preparation. This will help me to evaluate if there are any suggestions I can make to improve your results or if the antibodies are faulty.


I look forward to your reply so that I may assist you further. Please do not hesitate to contact me with any additional questions or concerns.

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Answer

Thank you for your feedback. I am happy to hear that the replacement vial provides good results. In order to increase the amount of tubulin that is IPed, I have a couple protocol tips that might be worth trying: 1.) Tubulin is a very abundant protein and you are using a high amount of tissue and 5ug of antibody might be saturated without catching all the tubulin. I therefore suggest to increase the amount of antibody. 2.) It is possible that the binding of bead-antibody and tubulin is not optimal. I suggest to increase the incubation times and to make sure that the samples are under gentle agitation or rotation. Since there are two possibilities to incubate: (A) beads and antibody first or (B) antibody and sample first, I suggest to try both to see if the binding can be optimized. 3.) I can also recommend to check if the buffers (if using naive buffer, make sure the protein is not denatured during the IP) and proteinase inhibitors are in working order to prevent denaturing or degenerating proteins and antibody. I hope this information is helpful. Please do not hesitate to contact me with any further questions in this matter.

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Answer

I am glad to hear the antibody is working well. (I'm not sure what happened to your original enquiry; we received an empty complaint form). A dilute stock solution will remain stable if you dilute the antibody you received in the buffer you use for preparing the working solution, for instance TBS or PBS, with the addition of BSA at a concentration of 1 mg/ml. The BSA minimizes antibody aggregation and loss to vessel adhesion. Do not include detergent. Aliquot and store at -20C. In general, the more concentrated the stock, the more stable it will be. Diluting 10-fold or 20-fold should be acceptable, but if diluting much more than that for stock storage, e.g., 100-fold, I would first test a sample of 100-fold-diluted stock, frozen and then thawed, by comparing the signal obtained from a test lane (cropped from a blot) to the signal from the original stock. Please let me know if you have any questions.

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Answer

Thank you for your enquiry. I tried to find any data about testing of TU-20 on rabbit tissue in papers, however I found only data about mouse, boar, porcine and human tissues. All tested species are specified on Abcam product datasheets, and these are updated as soon as any new information is brought to our attention. However, this does not mean that the antibody will not work, just that we have not tested it and therefore, can not guarantee that you will get good results. If you decide to go ahead and purchase this product, we encourage you to submit an Abreview via the online product datasheet. We always encourage customers to send their results back to us, whether positive or negative, and we make all product information available to researchers. We reward each Abreview with Abpoints which can be used for discounts off future purchases and gifts. To find out more about our Abreview system, please see the following URL: https://www.abcam.com/abreviews I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your enquiry. This antibody shows a band at around 52-56 kDa on blots. dependant on the conditions. Please let me know if I can be of any further assistance.

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1-10 of 15 Q&A

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