Overview

  • Product nameAnti-Tubulin antibody [YL1/2]
    See all Tubulin primary antibodies
  • Description
    Rat monoclonal [YL1/2] to Tubulin
  • SpecificityThis antibody detects the tyrosinated form of the alpha-tubulin subunit. It has equal affinity for fixed microtubules (formaldehyde or glutaraldehyde) and native microtubules. The antibody recognises an extremely short amino acid sequence Glu-Glu-Phe(OH) which can be inserted into the C-terminal domain of fusion proteins.
  • Tested applicationsELISA, IHC-Fr, IP, RIA, WB, Flow Cyt, ICC/IF, IHC (PFA fixed), IHC-P, IHC (Methanol fixed)more details
  • Species reactivity
    Reacts with: Mouse, Human, Pig, Saccharomyces cerevisiae, Xenopus laevis, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), Schizosaccharomyces pombe, African Green Monkey
    Predicted to work with: a wide range of other species, all Mammals
  • Immunogen

    Full length native protein (purified) (S. cerevisiae).

  • EpitopeA linear sequence requiring an aromatic residue at the C terminus, with the two adjacent amino acids being negatively charged (represented by Gly-Gly-Tyr in Tyr-Tubulin).
  • Positive control
    • This antibody gave a positive signal in HeLa, NIH 3T3 and PC12 whole cell lysates. In Flow Cytometry, this antibody gave a positive signal in methanol fixed/Tween permeabilised HeLa cells. IHC-P: FFPE human colon tissue sections.
  • General notesWe can conjugate this antibody to FITC for you (please see ab150204 for details).


    This antibody can be used as a loading control on Western blots (Allen et al.) and is not detected by anti-mouse Ig secondaries. It has been used in epitope tagging procedures to detect proteins tagged with a C-terminal Gly-Gly-Phe(OH) epitope. Under some circumstances this antibody may cross-react with other protein including E. coli rec A and oxidized actin.

Properties

Applications

Our Abpromise guarantee covers the use of ab6160 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
ELISA Use at an assay dependent dilution.
IHC-Fr Use at an assay dependent dilution.
IP Use at an assay dependent dilution.
RIA Use at an assay dependent dilution.
WB 1/5000 - 1/10000.
Flow Cyt Use 1µg for 106 cells.
ICC/IF 1/1000. (see PMID: 16230461)
IHC (PFA fixed) Use a concentration of 5 µg/ml. (from PubMed:16966421)
IHC-P Use at an assay dependent dilution.
IHC (Methanol fixed) 1/100. (from PubMed:16943269).

Target

  • RelevanceTubulin 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. Dimer of alpha and beta chains. A typical microtubule is a hollow water-filled tube with an outer diameter of 25 nm and an inner diameter of 15 nM. Alpha-beta heterodimers associate head-to-tail to form protofilaments running lengthwise along the microtubule wall with the beta-tubulin subunit facing the microtubule plus end conferring a structural polarity. Microtubules usually have 13 protofilaments but different protofilament numbers can be found in some organisms and specialized cells.
  • Cellular localizationCytoplasmic
  • Database links
    • Alternative names
      • Tubulin alpha-1 chain antibody
      • Tubulin alpha-3 chain antibody

    Anti-Tubulin antibody [YL1/2] images

    • IHC image of Tubulin staining in human colon formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ 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 ab6160, 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.

      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.

      *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

    • ICC/IF image of ab6160 stained HeLa cells. 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 ab6160 at 5µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rat (ab150165) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

    • All lanes : Anti-Tubulin antibody [YL1/2] (ab6160) at 1 µg/ml

      Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
      Lane 2 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
      Lane 3 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      Peroxidase Conjugated AffiniPure Rabbit Anti-Rat IgG (H+L) at 1/10000 dilution

      Performed under reducing conditions.

      Predicted band size : 50 kDa
      Observed band size : 52 kDa (why is the actual band size different from the predicted?)
      Additional bands at : 85 kDa. We are unsure as to the identity of these extra bands.

      Exposure time : 8 minutes
    • Lanes 1 & 3 : Anti-Tubulin antibody [YL1/2] (ab6160) at 1/5000 dilution
      Lanes 2 & 4 : Anti-Tubulin antibody [YL1/2] (ab6160) at 1/10000 dilution

      Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
      Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
      Lane 3 : BALB/3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab7901)
      Lane 4 : BALB/3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab7901)

      Lysates/proteins at 20 µg per lane.


      Predicted band size : 50 kDa
      Observed band size : 52 kDa (why is the actual band size different from the predicted?)
      Additional bands at : 17 kDa,34 kDa,80 kDa. We are unsure as to the identity of these extra bands.

      Exposure time : 10 seconds

      Western blot against tubulin with ab6160. Secondary Rabbit polyclonal to Rat IgG H&L (HRP) (ab6734) was used at 1/2000.

      Exposure time: 10 seconds.

      Lane 1-2: 20µg/lane HeLa (Human) whole cell lysate (ab7898)

      Lane 3-4: 20µg/lane 3T3 (Mouse) cell lysate (ab7901)

      Lane 1,3: ab6160 at 1/5000.

      Lane 2,4: ab6160 at 1/10000.

    • ab6160 staining Tubulin in mouse MEF cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed with 2% PFA  and 96% Ethanol. Samples were incubated with primary antibody (1/2000 in 0.1% Saponin/1% BSA/PBS) for 1 hour. A Cyt3®-conjugated goat polyclonal to rat IgG (H&L) was used at dilution at 1/500 as secondary antibody. Red staining in the image represents Tubulin, whereas the green one resembles gamma-tubulin.

      See Abreview

    • Overlay histogram showing HeLa cells stained with ab6160 (red line). The cells were fixed with 80% methanol (5 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 (ab6160, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rat IgG (H+L) (ab98386) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rat IgG2a [aRTK2758] (ab18450, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

    • This image was kindly supplied as part of the review submitted by Marko Kallio. Ab6160 was used for immunofluorescence on male rat testis samples in order to visualize microtubules of meiotically deviding cells. The samples were fixed with 2% paraformaldehyde and 0.8% glutaraldehyde and the antibody was used at a dilution 1:2500 (red - tubulin, blue - DNA stained with DAPI).
    • ab6160 staining mouse prostate tissue sections by IHC-P.  The tissue was serial sectioned at 6 microns, formaldehyde fixed and subjected to heat mediated antigen retrieval prior to blocking in 3% peroxidase for 5 minutes at 27°C.  The primary antibody was diluted 1/500 and incubated with the sample for 16 hours at 4°C.  A Cy5® conjugated goat anti-rat antibody was used as the secondary.

      See Abreview

    • ab6160 at 1/1000 dilution staining Tubulin in human WBC cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed in acetone and then blocked in 5% serum for 1 hour at 25°C. No permeabilization was done. The primary antibody was used at 1/1000 dilution in PBS-Tween and incubated with sample at 4°C for 16 hours. An Alexa Fluor® 594 conjugated goat polyclonal to rat IgG was used as secondary at 1/500 dilution.

      See Abreview

    • ab6160 at a 1/200 staining Tubulin in mouse liver tissue sections by Immunohistochemistry (frozen sections) incubated for 9 hours at +4°C. Fixed in formaldehyde, permeabilized using 0.2% Triton X-100. Blocked using 2% BSA for 30 minutes at 20°C. Secondary used at a 1/200 dilution polyclonal Goat anti-rat IgG conjugated to Alexa Fluor 555.

      See Abreview

    References for Anti-Tubulin antibody [YL1/2] (ab6160)

    This product has been referenced in:
    • Huang J  et al. Lutheran/basal cell adhesion molecule accelerates progression of crescentic glomerulonephritis in mice. Kidney Int N/A:N/A (2014). Mouse . Read more (PubMed: 24429403) »
    • Marno KM  et al. Novel restriction factor RNA-associated early-stage anti-viral factor (REAF) inhibits human and simian immunodeficiency viruses. Retrovirology 11:3 (2014). WB ; Human . Read more (PubMed: 24410916) »

    See all 78 Publications for this product

    Product Wall

    Application Immunocytochemistry/ Immunofluorescence
    Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
    Sample Zebrafish Cell (heart)
    Specification heart
    Permeabilization Yes - Acetone and Triton
    Fixative Formaldehyde
    Username

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    Verified customer

    Submitted Jun 20 2014

    Application Western blot
    Loading amount 50 µg
    Gel Running Conditions Reduced Denaturing (12% gel)
    Sample Mouse Tissue lysate - whole (Skeletal muscle)
    Specification Skeletal muscle
    Blocking step Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
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    Submitted Jan 08 2014

    Western blot

    Excellent
    Abreviews
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Western blot
    Loading amount 5 µg
    Gel Running Conditions Non-reduced Non-Denaturing (Native) (4-12% Bis-Tris Gel, MES running buffer)
    Sample Mouse Cell lysate - whole cell (Mouse whole brain tissue lysate, striatum and cort)
    Specification Mouse whole brain tissue lysate, striatum and cort
    Blocking step Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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    Submitted May 17 2013

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample Mouse Tissue sections (Mouse, brain tissue, whole brain sections, cortica)
    Specification Mouse, brain tissue, whole brain sections, cortica
    Fixative Paraformaldehyde
    Antigen retrieval step None
    Permeabilization Yes - Tween-20
    Blocking step Heat-inactivated normal donkey serum in 0.05% PBS-T as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
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    Submitted May 03 2013

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Immunohistochemistry (Frozen sections)
    Sample Mouse Tissue sections (E15.5 brain)
    Specification E15.5 brain
    Fixative Paraformaldehyde
    Permeabilization No
    Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
    Username

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    Submitted Nov 07 2012

    Thank you for contacting Abcam.

    We have 52 references that we know have used this antibody successfully, but I cannot find any that have used S.Pombe. I have however attached an image of our western blot testing that was done in house. The ant...

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    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Western blot
    Sample Human Cell lysate - whole cell (HeLa cells)
    Loading amount 15 µg
    Specification HeLa cells
    Gel Running Conditions Reduced Denaturing (10)
    Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
    Username

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    Verified customer

    Submitted Jun 08 2012

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Immunocytochemistry/ Immunofluorescence
    Sample Human Cell (HeLa cells)
    Specification HeLa cells
    Fixative Paraformaldehyde
    Permeabilization Yes - 0.5% Triton X100
    Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 37°C
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    Submitted Jun 08 2012

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Western blot
    Sample Human Cell lysate - whole cell (A549)
    Loading amount 20 µg
    Specification A549
    Treatment TRAIL
    Gel Running Conditions Reduced Denaturing (12.5% SDS-PAGE)
    Blocking step Odyssey buffer as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 100%
    Username

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    Submitted Apr 02 2012

    We now have new lot in stock so I can send you one vial as a replacement antibody. Again because we haven't tested this lot in mouse so we cannot confirm the ab will work.

    You have kindly mentioned in previous emails that the antibody worked ...

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