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

Overview

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 Abreviews Notes
Flow Cyt Use 1µg for 106 cells.



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

ICC/IF Use a concentration of 0.5 - 1 µg/ml.
IP Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Electron Microscopy Use at an assay dependent concentration.
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 antibody [DM1A] - Loading Control images

  • IHC image of ab7291 staining alpha Tubulin in rat colon 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 (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab7291, 0.5ug/ml working concentration, 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. 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.

  • IHC image of ab7291 staining alpha Tubulin in human colon 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 (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab7291, 5ug/ml working concentration, 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. 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.
    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • FABP4 (green) was detected using FABP4 primary antibody (ab92501; diluted 1/1000). Alpha tubulin (red) was detected using the mouse monoclonal (ab7291) antibody. Cells were imaged by confocal microscopy, using z-stack for adipocyte-like cells.

  • ab7291 staining alpha Tubulin in SV40 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes 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 ab7291 at a working concentration of 0.5μg/ml and ab190573, Rabbit monoclonal [EP1332Y] to alpha Tubulin (Alexa Fluor® 647, shown in red) at 1/250 overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-mouse AlexaFluor® 488 (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

    This product also gave a positive signal in 100% methanol (5 min) fixed SV40 cells under the same testing conditions.

    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • ab7291 staining alpha-Tubulin in Caco-2 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 ab7291 at 1μg/ml and ab6046 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-mouse AlexaFluor® 488 (ab150117) at 2 μg/ml (shown in green) and anti-rabbit AlexaFluor® 594 (ab150088) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

    Negative controls: 1– Rabbit primary antibody 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.

  • ab7291 staining alpha Tubulin in NIH3T3 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 ab7291 at 1μl/ml and ab6046 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-mouse AlexaFluor® 488 (ab150117) at 2 μg/ml (shown in green) and anti-rabbit AlexaFluor® 594 (ab150088) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

    Negative controls: 1– Rabbit primary antibody 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-alpha Tubulin antibody [DM1A] - 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 Anti-Mouse Alexa Fluor® 790 (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 antibody [DM1A] - 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 HRP (ab97040), 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 antibody [DM1A] - 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

    Secondary: anti-mouse HRP (ab6789)

  • 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 an anti-mouse DyLight® 488 (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.

    Secondary: anti-mouse HRP (ab6728)

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

This product has been referenced in:
  • Gutiérrez-Caballero C  et al. TACC3-ch-TOG track the growing tips of microtubules independently of clathrin and Aurora-A phosphorylation. Biol Open N/A:N/A (2015). WB . Read more (PubMed: 25596274) »
  • Khare V  et al. Overexpression of PAK1 promotes cell survival in inflammatory bowel diseases and colitis-associated cancer. Inflamm Bowel Dis 21:287-96 (2015). Read more (PubMed: 25569743) »

See all 135 Publications for this product

Product Wall

Application Immunocytochemistry/ Immunofluorescence
Sample Pig Cell (PK15)
Permeabilization Yes - 1% Triton X-100, 5 mins, Room Temp
Specification PK15
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 21°C
Fixative Paraformaldehyde
Username

Dr. Rob Noad

Verified customer

Submitted Jul 17 2015

Application Western blot
Loading amount 50 µg
Gel Running Conditions Reduced Denaturing (12% polyacrylamide)
Sample Human Cell lysate - whole cell (Huh7.5 hepatoma cell line)
Specification Huh7.5 hepatoma cell line
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Anthony Morel

Verified customer

Submitted Nov 10 2014

Application Western blot
Loading amount 40 µg
Gel Running Conditions Reduced Denaturing (10% SDS PAGE)
Sample Mouse Tissue lysate - whole (Liver)
Specification Liver
Blocking step Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jul 02 2014

Application Western blot
Loading amount 1.2e+006 cells
Gel Running Conditions Reduced Denaturing
Sample Rat Cell lysate - whole cell (Insulinoma Cell Line (INS-1))
Specification Insulinoma Cell Line (INS-1)
Blocking step Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Username

Dr. Javier Triñanes

Verified customer

Submitted Jun 04 2014

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
Username

Abcam user community

Verified customer

Submitted Mar 28 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
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
Username

Abcam user community

Verified customer

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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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)
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
Username

Abcam user community

Verified customer

Submitted Apr 23 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
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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