Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)
- Product nameAnti-alpha Tubulin antibody [DM1A] - Loading ControlSee all alpha Tubulin primary antibodies ...
- DescriptionMouse 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 reactivityReacts with: Mouse, Rat, Chicken, Guinea pig, Hamster, Cow, Dog, Human, Pig, Xenopus laevis, Gerbil, African Green Monkey
Full length native protein (purified) of Chicken alpha Tubulin (extracted from brain).
- Epitopeaa 426-450
- Positive controlIn Flow Cytometry, this antibody gave a positive signal in methanol fixed/Tween permeabilised HeLa cells.
- General notes
Excellent as a protein loading control antibody.
- Storage instructionsStore at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
- Storage bufferPreservative: 0.02% Sodium Azide
- Concentration information loading...
- PurityIgG fraction
- Primary antibody notes Excellent as a protein loading control antibody.
- Clonality Monoclonal
- Clone numberDM1A
- Light chain typekappa
- Research Areas
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.
|Flow Cyt||Flow Cyt: Use 1µg for 106 cells.|
|ICC/IF||ICC/IF: Use a concentration of 1 µg/ml.|
|IP||IP: Use at an assay dependent dilution.|
|IHC-P||IHC-P: Use at an assay dependent dilution. Antigen retrieval is not essential but may optimise staining.|
|IHC-Fr||IHC-Fr: Use at an assay dependent concentration.|
|Electron Microscopy||EM: Use at an assay dependent dilution.|
|WB||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.|
- 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
- Entrez Gene: 429035 Chicken
- Entrez Gene: 504244 Cow
- Entrez Gene: 7277 Human
- Entrez Gene: 7846 Human
- Entrez Gene: 7278 Human
- Entrez Gene: 22142 Mouse
- Entrez Gene: 64158 Rat
- Entrez Gene: 399313 Xenopus laevis
- Omim: 191110 Human
- SwissProt: Q5E986 Cow
- SwissProt: P68366 Human
- SwissProt: Q71U36 Human
- SwissProt: Q13748 Human
- SwissProt: P68369 Mouse
- SwissProt: P68370 Rat
- SwissProt: Q4V850 Xenopus laevis
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Anti-alpha Tubulin antibody [DM1A] - Loading Control images
Predicted band size : 50 kDa
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
Goat polyclonal Secondary Antibody to 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.
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.
References for Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)
This product has been referenced in:
- Jin L et al. Ubiquitin-dependent regulation of COPII coat size and function. Nature 482:495-500 (2012). Read more (PubMed: 22358839) »
- Ho HY et al. Wnt5a-Ror-Dishevelled signaling constitutes a core developmental pathway that controls tissue morphogenesis. Proc Natl Acad Sci U S A 109:4044-51 (2012). Read more (PubMed: 22343533) »