Anti-alpha Tubulin [DM1A] antibody - Loading Control (ab7291)

Overview

  • Product nameAnti-alpha Tubulin [DM1A] antibody - Loading ControlSee all alpha Tubulin primary antibodies ...
  • Description
    Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control
  • SpecificityDM1A causes the 10 nm filaments to collapse into large lateral aggregates collecting in the cell periphery or tight juxtanuclear caps. It does not block microtubule assembly. It does not inhibit polymerisation or depolymerisation of platelet tubulin in vitro. It blocks (by 70-80%) the ability of tubulin dimers (with GppNHp bound) to promote a stable inhibition of adenylyl cyclase. See references for further information on the above.
  • Tested applicationsFlow Cyt, ICC/IF, IP, IHC-P, IHC-Fr, Electron Microscopy, WB more details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Guinea pig, Hamster, Cow, Dog, Human, Pig, Xenopus laevis, Gerbil, African Green Monkey
  • Immunogen

    Full length native protein (purified) of Chicken alpha Tubulin (extracted from brain).

  • Epitopeaa 426-450
  • Positive control
    • This antibody gave a positive signal within WB in the following whole cell lysates: HeLa; HEK293; HepG2; Caco2; NIH3T3; PC12. In Flow Cytometry, this antibody gave a positive signal in methanol fixed/Tween permeabilised HeLa cells.
  • General notes


    Excellent as a protein loading control antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab7291 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
Flow Cyt Use 1µg for 106 cells.
ICC/IF Use a concentration of 1 µg/ml.
IP Use at an assay dependent dilution.
IHC-P Use at an assay dependent dilution. Antigen retrieval is not essential but may optimise staining.
IHC-Fr Use at an assay dependent concentration.
Electron Microscopy Use at an assay dependent dilution.
WB 1/5000 - 1/10000. Detects a band of approximately 50 kDa (predicted molecular weight: 50 kDa). Lower background can be obtained by diluting ab7291 to 1:10000 and incubating overnight at 4C (Customer communication).This antibody is ideal for use as a Western blot loading control.Works under both reducing and non-reducing conditions.

Target

  • RelevanceMicrotubules are involved in a wide variety of cellular activities ranging from mitosis and transport events to cell movement and the maintenance of cell shape. Tubulin itself is a globular protein which consists of two polypeptides (alpha and beta tubulin). Alpha and beta tubulin dimers are assembled to 13 protofilaments that form a microtubule of 22 nm diameter. Tyrosine ligase adds a C-terminal tyrosin to monomeric alpha tubulin. Assembled microtubules can again be detyrosinated by a cytoskeleton associated carboxypeptidase. Detyrosinated alpha tubulin is referred to as Glu-tubulin. Another post-translational modification of detyrosinated alpha tubulin is C-terminal polyglutamylation which is characteristic for microtubules in neuronal cells and the mitotic spindle. Alpha tubulin is not suitable as a loading control in adipose tissue as expression of tubulin in adipose tissue is very low ( Spiegelman and Farmer, Cell, 1982, 29(1): 53-60, "in cells undergoing adipose differentiation actin synthesis decreases by 90%").
  • Cellular localizationMajor constituent of Microtubules
  • Database links
  • Alternative names
    • Alpha-tubulin 1 antibody
    • FLJ30169 antibody
    • H2 ALPHA antibody
    • Testis specific alpha tubulin antibody
    • TUBA1 antibody
    • TUBA1A antibody
    • TUBA2 antibody
    • TUBA3 antibody
    • TUBA3C antibody
    • TUBA4A antibody
    • Tubulin alpha 1 chain antibody
    • Tubulin H2 alpha antibody
    • Tubulin, alpha 1a antibody
    • Tubulin, alpha 3c antibody
    • Tubulin, alpha 4a antibody
    see all

Anti-alpha Tubulin [DM1A] antibody - Loading Control images

  • All lanes : Anti-alpha Tubulin [DM1A] antibody - Loading Control (ab7291) at 1/5000 dilution

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
    Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
    Lane 4 : Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate
    Lane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
    Lane 6 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat Anti-Mouse IgG H&L (Alexa Fluor® 790) (ab175783) at 1/10000 dilution

    Predicted band size : 50 kDa
    Observed band size : 52 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 Licor blocking buffer before being incubated with ab7291 overnight at 4°C. Antibody binding was detected using ab175783 at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

  • All lanes : Anti-alpha Tubulin [DM1A] antibody - Loading Control (ab7291) 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
    Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/50000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 50 kDa
    Observed band size : 50 kDa


    Exposure time : 150 seconds

    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 2% Bovine Serum Albumin before being incubated with ab7291 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution ab133406

  • ICC/IF image of ab7291 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab7291, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

  • Immunofluorescent imaging of human cells (U2OS) with ab7291 reveals a delicate network of alpha-tubulin (green) located exclusively in the cytoplasm. The nucleus is stained blue.

    IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour.  Primary antibody used at 1/200 in 5% milk / 0.2% TWEEN for one hour, secondary antibody Alexa 488 for 30 minutes.  All blocking and incubation steps carried out at 37 degrees.

    Unfortunately, due to size constraints for images on our website, we are unable to show the full uncompressed picture in all its glory!

  • Anti-alpha Tubulin [DM1A] antibody - Loading Control (ab7291) at 0.5 µg/ml + Hela cell lysate

    Secondary
    Goat Anti-Mouse IgG H&L (HRP) (ab6789) at 1/5000 dilution
    developed using the ECL technique

    Performed under non-reducing conditions.

    Predicted band size : 50 kDa
    Observed band size : 57 kDa (why is the actual band size different from the predicted?)


    Exposure time : 30 seconds
  • ab7291 at 1/400 staining MCF-10A cells (human mammary epithelial cell line) by ICC/IF. The cells were methanol fixed, blocked with BSA and incubated with the antibody for 16 hours. An Alexa-Fluor ® 488 conjugated goat anti-mouse antibody was used as the secondary. The image shows alpha tubulin staining in green and DAPI counterstain in blue.

    See Abreview

  • Overlay histogram showing HeLa cells stained with ab7291 (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 (ab7291, 1µg/1x106 cells) 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.

  • IF image of ab7291 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 (ab7291, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 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. This antibody also gave a positive result in 100% methanol fixed (5 min) Hek293, HepG2 and MCF7 cells at 1µg/ml, and in 4% formaldehyde fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.


  • Predicted band size : 50 kDa

    Western blot using ab7291 on Hela Whole Cell Lysate (20µg/lane).

    Exposure: 10 sec.
    Secondary: ab6728.

    Lane 1: ab7291 at 1/5000
    Lane 2: ab7291 at 1/10000.

References for Anti-alpha Tubulin [DM1A] antibody - Loading Control (ab7291)

This product has been referenced in:
  • Rana T  et al. Complement Protective Epitopes and CD55-Microtubule Complexes Facilitate the Invasion and Intracellular Persistence of Uropathogenic Escherichia coli. J Infect Dis 209:1066-76 (2014). Chinese Hamster . Read more (PubMed: 24259524) »
  • Shrivastava V  et al. Cigarette smoke affects posttranslational modifications and inhibits capacitation-induced changes in human sperm proteins. Reprod Toxicol 43:125-9 (2014). Read more (PubMed: 24345728) »

See all 90 Publications for this product

Product Wall

Application Western blot
Loading amount 1e+006 cells
Gel Running Conditions Reduced Denaturing (10%)
Sample Mouse Cell lysate - whole cell (mouse ES cells)
Specification mouse ES cells
Blocking step Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
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Verified customer

Submitted Mar 28 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Sample Human Cell (human epithelial cells)
Specification human epithelial cells
Permeabilization Yes - 0.1% triton X 100
Fixative Formaldehyde
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Submitted Jul 01 2013



Ich habe drei Antikörper gefunden, die nicht aus Maus stammen und für un-reduzierte Proben geeignet scheinen. Dies haben wir nicht selbst getestet, sondern diese Information stammt von anderen Kunden, die Abreviews eingereicht haben...

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Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (Mouse, brain tissue, whole brain sections)
Specification Mouse, brain tissue, whole brain sections
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 Apr 23 2013

Application Western blot
Sample Human Cell lysate - whole cell (Daudi (Human Burkitt's lymphoma cell line))
Loading amount 20 µg
Specification Daudi (Human Burkitt's lymphoma cell line)
Gel Running Conditions Reduced Denaturing (12%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Username

Dr. Elena Kashuba

Verified customer

Submitted Apr 05 2013

There are several different alpha Tubulin genes (alpha-1 to alpha-8). When the human sequence (aa426-448) is BLASTed against chicken it was most similar to the chicken alpha-5 and alpha-4 proteins which are both 448+ AA long. Our lab therefore suspects...

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Application Immunocytochemistry/ Immunofluorescence
Sample Monkey Cell (FRhK-4 cells)
Specification FRhK-4 cells
Fixative Paraformaldehyde
Permeabilization Yes - 0.2%Triton-X100 in PBS
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 37°C
Username

Abcam user community

Verified customer

Submitted Nov 07 2012

Thank you for your inquiry. I am happy to confirm that all these products are suitable for WB and tested and guaranteed.
I suggest ab29889 (Liver (Human) Tissue Lysate - adult normal tissue) as positive control for ab3366, ab3373, ab3375 and ab241...

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Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (U2OS)
Specification U2OS
Fixative Formaldehyde
Permeabilization Yes - Triton 0,5% for 5 min
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
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Verified customer

Submitted Jan 25 2012

Application Western blot
Sample Human Tissue lysate - whole (Adipose tissue)
Loading amount 20 µg
Specification Adipose tissue
Gel Running Conditions Reduced Denaturing (10 %)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Mrs. Maria Insenser

Verified customer

Submitted Jan 24 2012

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"