Anti-beta Tubulin antibody - Loading Control (ab6046)

Rabbit polyclonal beta Tubulin antibody. Validated in WB, IP, ELISA, IHC, ICC, ICC/IF and tested in Mouse, Rat, Chicken, Human, Pig, Xenopus laevis, Zebrafish, Chinese hamster.

Overview

  • Product name
    Anti-beta Tubulin antibody - Loading Control
    See all beta Tubulin primary antibodies
  • Description
    Rabbit polyclonal to beta Tubulin - Loading Control
  • Host species
    Rabbit
  • Specificity
    This antibody detects a single clean band at 50kD representing beta Tubulin. This band is significantly reduced by using peptide blocking.
  • Tested applications
    Suitable for: ICC, IHC-Fr, WB, ICC/IF, ELISA, IHC-P, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Human, Pig, Xenopus laevis, Zebrafish, Chinese hamster
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human beta Tubulin.

    Read Abcam's proprietary immunogen policy (Peptide available as ab20775.)

  • Positive control
    • HeLa Cell lysate; A431 Cell lysate; MCF7 Cell lysate; 293 Cell lysate; HeLa Cell lysate; A431 Cell lysate; MCF7 Cell lysate; 293 Cell lysate;
  • General notes

    Abcam recommended secondaries - Goat Anti-Rabbit HRP (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077).

    See other anti-rabbit secondary antibodies that can be used with this antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab6046 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 1/200. (see Abreview)
IHC-Fr 1/200. (see Abreview)
WB 1/500. Detects a band of approximately 50 kDa (predicted molecular weight: 50 kDa).
ICC/IF 1/200. (see PMID: 16030258)
ELISA Use at an assay dependent concentration.
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IP Use at an assay dependent concentration.

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.
  • Tissue specificity
    Ubiquitously expressed with highest levels in spleen, thymus and immature brain.
  • Involvement in disease
    Cortical dysplasia, complex, with other brain malformations 6
    Skin creases, congenital symmetric circumferential, 1
  • 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, resulting in polyglutamate chains on the gamma-carboxyl group (PubMed:26875866). Polyglutamylation plays a key role in microtubule severing by spastin (SPAST). SPAST preferentially recognizes and acts on microtubules decorated with short polyglutamate tails: severing activity by SPAST increases as the number of glutamates per tubulin rises from one to eight, but decreases beyond this glutamylation threshold (PubMed:26875866).
    Some glutamate residues at the C-terminus are 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). Both polyglutamylation and monoglycylation can coexist on the same protein on adjacent residues, and lowering glycylation levels increases polyglutamylation, and reciprocally. The precise function of monoglycylation is still unclear.
    Phosphorylated on Ser-172 by CDK1 during the cell cycle, from metaphase to telophase, but not in interphase. This phosphorylation inhibits tubulin incorporation into microtubules.
  • Cellular localization
    Cytoplasm, cytoskeleton.
  • Information by UniProt
  • Database links
  • Alternative names
    • Beta 4 tubulin antibody
    • Beta 5 tubulin antibody
    • beta Ib tubulin antibody
    • Beta1 tubulin antibody
    • Class I beta tubulin antibody
    • M40 antibody
    • MGC117247 antibody
    • MGC16435 antibody
    • OK/SW cl.56 antibody
    • OK/SWcl.56 antibody
    • TBB5_HUMAN antibody
    • TUBB 1 antibody
    • TUBB 2 antibody
    • TUBB 5 antibody
    • TUBB antibody
    • TUBB1 antibody
    • TUBB2 antibody
    • TUBB5 antibody
    • tubulin beta 1 chain antibody
    • Tubulin beta 2 chain antibody
    • tubulin beta 5 chain antibody
    • Tubulin beta chain antibody
    • Tubulin beta class I antibody
    • tubulin beta polypeptide antibody
    • Tubulin beta-5 chain antibody
    see all

Images

  • Lane 1 - 8 : beta Tubulin antibody - Loading Control (ab6046) at 1/500 dilution

    Lane 1 : HeLa Cell lysate at 20 ug
    Lane 2 : A431 Cell lysate at 20 ug
    Lane 3 : MCF7 Cell lysate at 20 ug
    Lane 4 : 293 Cell lysate at 20 ug with beta Tubulin peptide (ab20775) at 1 ug/ml
    Lane 5 : HeLa Cell lysate at 20 ug with beta Tubulin peptide (ab20775) at 1 ug/ml
    Lane 6 : A431 Cell lysate at 20 ug with beta Tubulin peptide (ab20775) at 1 ug/ml
    Lane 7 : MCF7 Cell lysate at 20 ug with beta Tubulin peptide (ab20775) at 1 ug/ml
    Lane 8 : 293 Cell lysate at 20 ug with beta Tubulin peptide (ab20775) at 1 ug/ml

  • ICC/IF image of ab6046 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6046, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab150081 Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

    The negative control (inset) is a secondary-only assay to demonstrate low non-specific binding of the secondary antibody.

    This product also gave a positive signal under the same testing conditions in HeLa cells fixed with 100% methanol (5 min).

  • ab6046 staining beta Tubulin in human stomach tissue by Immunohistochemistry (frozen sections). Tissue was fixed with acetone and then blocked with 5% serum for 1 hour at 23°C followed by incubation with the primary antibody at a 1/200 dilution for 1 hour at 23°C. An undiluted HRP-conjugated goat polyclonal was used as secondary antibody.

    See Abreview

  • ICC/IF image of ab6046 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6046, 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Adiponectin (green) was detected using adiponectin primary antibody (ab22554; 2.5 µl/mL). Beta tubulin (red) was detected using the rabbit polyclonal (ab6046) antibody. Cells were imaged by confocal microscopy, using z-stack for adipocyte-like cells.

  • Beta Tubulin was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Tubulin and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab6046.
    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
    Band: 50kDa: beta Tubulin.
  • The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6046, 1µg/ml and ab37266, 1µg/ml) overnight at +4°C. The secondary antibodies were ab150115 Alexa Fluor® 647 goat anti-mouse IgG (H+L) used at 2µg/ml for 1h and ab175652 Alexa Fluor® 405 goat anti-rabbit IgG (H+L) used at 2µg/ml for 1h. Nuclear Green LCS1 (ab138904) was used to stain the cell nuclei (green) at a dilution of 1/500.

     

  • IHC image of beta Tubulin staining in human liver carcinoma FFPE section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab6046, 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 haematoxylin and mounted with DPX.
  • Lane 1 - 8 : beta Tubulin antibody - Loading Control (ab6046) at 1/500 dilution

    Lane 1 : HeLa Cell lysate at 20 ug
    Lane 2 : A431 Cell lysate at 20 ug
    Lane 3 : MCF7 Cell lysate at 20 ug
    Lane 4 : 293 Cell lysate at 20 ug with beta Tubulin peptide (ab20775) at 1 ug/ml
    Lane 5 : HeLa Cell lysate at 20 ug with beta Tubulin peptide (ab20775) at 1 ug/ml
    Lane 6 : A431 Cell lysate at 20 ug with beta Tubulin peptide (ab20775) at 1 ug/ml
    Lane 7 : MCF7 Cell lysate at 20 ug with beta Tubulin peptide (ab20775) at 1 ug/ml
    Lane 8 : 293 Cell lysate at 20 ug with beta Tubulin peptide (ab20775) at 1 ug/ml

    Secondary

References

This product has been referenced in:
  • de Sousa FD  et al. Hydrogel and membrane scaffold formulations of Frutalin (breadfruit lectin) within a polysaccharide galactomannan matrix have potential for wound healing. Int J Biol Macromol 121:429-442 (2019). Read more (PubMed: 30326222) »
  • Zhang S  et al. Targeting PRMT5/Akt signalling axis prevents human lung cancer cell growth. J Cell Mol Med 23:1333-1342 (2019). Read more (PubMed: 30461193) »
See all 520 Publications for this product

Customer reviews and Q&As

1-10 of 18 Q&A

Question
Answer

We would recommend the use of one of the following products for a loading control with zebrafish samples: AB15224 anti alpha Tubulin (https://www.abcam.com/alpha-tubulin-antibody-ab15246.html). This product has been featured in the following publication for use in WB on zebrafish (PubMed ID: 22581286). AB6046 anti beta Tubulin. This product has an excellent Abreview provided by a customer using in WB on zebrafish. This review is located at the following web address: https://www.abcam.com/beta-Tubulin-antibody-Loading-Control-ab6046-reviews.html?intabreviewid=15893 AB14744 anti- COX IV antibody (https://www.abcam.com/COX-IV-antibody-20E8C12-Mitochondrial-Loading-Control-ab14744-references.html). Another guaranteed product for use as loading control this product has been used in publication (PubMed ID: 18765295).

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Question

Today I ordered 100 ug of an anti- beta tubulin primary antibody. I received the antibody this afternoon (and I am grateful that I received it so quickly!). I briefly spun down the tube to get all of the antibody solution to the bottom of the tube. It should have contained 100 microliters in the tube. As I was putting aside aliquots to freeze at -20 degrees (in 10 microliter aliquots), the last tube had less than 5 microliters in it, instead of 10. Unfortunately, this precludes me from doing one western blot, which requires 1:500 dilution. Considering that the antibody cost over $300, this is approximately $30 that I spent and cannot use (10 microliters antibody is needed per western blot). I was wondering if its somehow possible to get another 10 microliters of the antibody so that I can complete the experiments which I had planned? This is a particularly important antibody for our lab, as it will serve as a loading control for all of our western blots. We often buy antibodies from Abcam and will plan on buying more in the future. However, at this point, it is not reasonable to buy another tube to get enough antibody for the 10th blot. Thank you, Jenny Linnoila, M.D., Ph.D. The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail.

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Answer

Thank you for contacting us. I am sorry that the vial you received did not contain the full amount of the product and I apologize for the inconvenience. I have issued a free of charge replacement vial with the order number of xxxx. I have asked this to be shipped to you on Tuesday because of the holiday weekend.. Please do not hesitate to contact us if you need anything further.

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Answer

Thank you for contacting us.

A) The concentration for this antibody is batch dependent. It may vary within the range 0.9-1mg/ml. We provide 100ug of antibody, this means the volume supplied may vary from 100-111uL.

B) To prepare a 1/500 dilution you can take, for instance, 1uL of antibody and take it to 500uL of antibody diluent (that is, add 499uL).

I hope this helps and if we can assist further, please do not hesitate to contact us.

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Answer

Thank you for contacting us.

The homology of the human immunogen peptide for ab6046 andab21057 is 84%. These antibodies are polyclonal.
For ab78109, which was raised against the pig protein, the homology of the full length protein is 86%. For ab40862, which was raised against the rat protein, the homology of the full length rat protein with T. brucei is also 86%. The latter 2 antibodies are monoclonal, but the binding site has not been determined.

Any of these 4 antibodies would be good to try, and you would be eligible for our testing discount program.

For UNTESTED species and/or applications, we have established a testing discount program. Here is a brief description of how it works:
The testing discount program is for customers who like to use an antibody/protein/kit on an untested species/application. You would purchase the antibody/protein/kit at full price, test it and submit an Abreview with your data (positive or negative). On your next order you will receive a discount for ONE antibody/protein at the full price (100%) of the antibody/protein you have tested, or a full price discount for the amount paid for the kit. The terms and conditions applicable to this offer can be found here: https://www.abcam.com/collaborationdiscount.
This programapplies to ELISA kits and other kits we offer.

Please let me know which antibody you'd like to try and I'd be happytosend you the discount code.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you for contacting us.

My recommendations are below. For a loading control for whole cell extracts, you could use any one of these, since the whole cell extract will contain all of these proteins. I have listed two proteins for the cytoplasmic fraction (GAPDH and tubulin). Both are appropriate but one may be preferable, depending on the size of your protein of interest.

nuclear fraction
Anti-TATA BP - ab818

cytoplasmic fraction
Anti-GAPDH - ab9485
Anti-tubulin - ab6046

whole cell extract
Anti-GAPDH - ab9485
Anti-tubulin - ab6046
Anti-TATBP - ab818

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Answer

Thank you for your response.
This is to let you know that I have placed a new order for you - for one vial of ab6046 as a free of charge replacement (from a new batch: GR73104-1) and the new order number is 1044538.
I hope the second vial will work as it is expected, and please do let me know how you are getting on with this product.
I would suggest using 5% BSA in TBS-T as blocking agent and diluting the primary and the secondary antibody in this solution. My other advice would be to use (if it is possible) Nonidet-P40 (NP40) buffer which contains high concentration of detergent to make sure that the proteins are properly extracted from the brain (which contains high levels of lipids and phospholipids).
Nonidet-P40 (NP40) buffer:
150 mM sodium chloride
1.0% NP-40 (Triton X-100 can be substituted for NP-40)
50 mM Tris, pH 8.0
You can find some useful details at this site:
https://www.abcam.com/index.html?pageconfig=resource&rid=11379#A1
If you need any further assistance in the future, please do not hesitate to contact me.

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Answer

Thank you for getting back to me and for confirming the batch and order numbers.
Blocking agent can cause different results. We have some lab data indicating that with certain antibodies used for loading control 5% BSA works much better than milk and this may be worth considering.
I could certainly offer you a new vial from a different batch if you wish to test it, or raise a credit note which you can use in the future.
Please do let me know how you wish to proceed. I look forward to hearing from you soon.

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Answer

Thank you for your enquiry regarding ab6046 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.
As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.
- Could you please confirm the batch number and the Abcam Order number?
- How was the blocking carried out? Was BSA or milk or other agent used?
I look forward to hearing from you and hope to solve this problem as soon as possible.

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Answer

Thank you for your enquiry and your interest. I regret to inform you that we are unable to change the format of these two particular antibodies; it is part of the normal production procedure. Apologies! If you need any further assistance in the future, please do not hesitate to contact me.    

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Answer

Thank you for contacting Abcam. I have searched our catalogue and found two antibodies which I believe will work for your experiments. The nuclear fraction loading control I would use is ab23474 which is a rabbit polyclonal to TATA binding protein. The other antibody I would recommend, for the cytoplasmic fraction is the ab8229, this is a goat polyclonal antibody to beta-actin. I hope that you find this information helpful. Please feel free to contact us again should you have further questions about these antibodies or any of the products in our catalogue.

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1-10 of 18 Q&A

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