製品の概要

  • 製品名

    Anti-alpha Tubulin antibody [DM1A] - Loading Control
    alpha Tubulin 一次抗体 製品一覧
  • 製品の詳細

    Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control
  • 由来種

    Mouse
  • 特異性

    Excellent as a protein loading control antibody. DM1A 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.
  • アプリケーション

    適用あり: Flow Cyt, ICC/IF, IP, IHC-Fr, IHC-P, Electron Microscopy, WBmore details
  • 種交差性

    交差種: Mouse, Rat, Chicken, Guinea pig, Hamster, Cow, Dog, Human, Pig, Xenopus laevis, Gerbil, African green monkey
  • 免疫原

    Full length native protein (purified) corresponding to Chicken alpha Tubulin.

  • エピトープ

    aa 426-450
  • ポジティブ・コントロール

    • WB: HeLa, HEK293, HepG2, Caco2, NIH3T3, PC12. Flow Cytometry: methanol fixed/Tween permeabilised HeLa cells. ICC/IF: Caco-2, NIH3T3, SV40LT-SMC. IHC-P: Human colon, Rat colon.
  • 特記事項

    This antibody clone [DM1A] is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

製品の特性

アプリケーション

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.

アプリケーション Abreviews 特記事項
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).

We recommend diluting ab7291 to 1:10000 and incubating overnight at 4°C. Works under both reducing and non-reducing conditions. We recommend using 3% BSA as the blocking agent, blocking with milk may cause a reduction in signal intensity.

ターゲット情報

  • 機能

    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.
  • 配列類似性

    Belongs to the tubulin family.
  • 翻訳後修飾

    Some glutamate residues at the C-terminus are polyglutamylated. This modification occurs exclusively on glutamate residues and results in polyglutamate chains on the gamma-carboxyl group. Also 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) whereas glutamylation is prevalent in neuronal cells, centrioles, axonemes, and the mitotic spindle. Both modifications can coexist on the same protein on adjacent residues, and lowering glycylation levels increases polyglutamylation, and reciprocally. The precise function of such modifications is still unclear but they regulate the assembly and dynamics of axonemal microtubules.
    Acetylation of alpha chains at Lys-40 stabilizes microtubules and affects affinity and processivity of microtubule motors. This modification has a role in multiple cellular functions, ranging from cell motility, cell cycle progression or cell differentiation to intracellular trafficking and signaling.
  • 細胞内局在

    Cytoplasm > cytoskeleton.
  • Information by UniProt
  • 参照データベース

  • 別名

    • Alpha-tubulin 1 antibody
    • ALS22 antibody
    • B ALPHA 1 antibody
    • bA408E5.3 antibody
    • H2 ALPHA antibody
    • Hum a tub1 antibody
    • Hum a tub2 antibody
    • LIS3 antibody
    • MGC171407 antibody
    • MGC55332 antibody
    • TBA4A_HUMAN antibody
    • Testis-specific alpha-tubulin antibody
    • TUBA1 antibody
    • TUBA1A antibody
    • tuba1l antibody
    • Tuba4a antibody
    • Tubulin alpha 1 chain antibody
    • Tubulin alpha antibody
    • Tubulin alpha-1 chain antibody
    • tubulin alpha-1B chain antibody
    • Tubulin alpha-4A chain antibody
    • Tubulin H2-alpha antibody
    • Tubulin, alpha 1 (testis specific) antibody
    • tubulin, alpha 1, like antibody
    • Tubulin, alpha 4a antibody
    • Tubulin, alpha, testis-specific antibody
    • Tubulin, alpha-1 antibody
    see all

画像

  • All lanes : Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution

    Lane 1 : HeLa 20ug
    Lane 2 : PC12 20ug
    Lane 3 : SV40LT-SMC 20ug
    Lane 4 : NIH 3T3 20ug
    Lane 5 : Rat liver 20ug
    Lane 6 : Rat heart 20ug

    Secondary
    All lanes : Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) at 1/20000 dilution

    Performed under reducing conditions.

    Predicted band size: 50 kDa



    Merged signal (red and green). Green - ab7291 observed at 52 kDa. Red - loading control, ab181602, observed at 38 kDa.

    All samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. Ab7291 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1/1,000 and 1/20,000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • Ab7291 staining alpha tubulin in human breast cancer cell line by ICC/IF (Immunocytochemistry/Immunofluorescence). Cells were fixed 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS  and blocked with 2% bovine serum albumin in sodium phosphate buffer. Cells were co-stained with anti-pericentrin using ab4448 at 1:500 dilution and ab7291 at 1:500 dilution. Alexa Fluor® 633 goat anti mouse and Alexa Fluor® 488 goat anti-rabbit (1:500 dilution) was used as secondary antibodies. DAPI was used as a nuclei counterstain. Representative images of mitotic cells with bipolar or multipolar spindles.

  • ab7291 staining alpha Tubulin in SV40LT-SMC 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).

  • 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

  • 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.

  • 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.

  • Lanes 2-7 : Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/2500 dilution

    Lane 1 : Marker
    Lanes 2-3 : Daudi (Human Burkitt's lymphoma cell line) at 10 µg
    Lanes 4-5 : Daudi (Human Burkitt's lymphoma cell line) at 15 µg
    Lanes 6-7 : Daudi (Human Burkitt's lymphoma cell line) at 20 µg

    Secondary
    Lanes 2-7 : HRP conjugated monoclonal Goat Anti-Mouse IgG at 1/1000 dilution

    Performed under reducing conditions.

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


    Exposure time: 1 minute

    See Abreview

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

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

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : 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

  • 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.

  • 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.

  • 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!

参考文献

This product has been referenced in:

  • Kline DD  et al. TRPV1 channels contribute to spontaneous glutamate release in nucleus tractus solitarii following chronic intermittent hypoxia. J Neurophysiol 121:881-892 (2019). Read more (PubMed: 30601692) »
  • Duarte FCK  et al. Association between naturally occurring spine osteoarthritis in geriatric rats and neurogenic inflammation within neurosegmentally linked skeletal muscle. Exp Gerontol 118:31-38 (2019). Read more (PubMed: 30615897) »
See all 521 Publications for this product

レビューと Q&A

1-10 of 20 Q&A

Question
Answer



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.

Bitte sehen Sie sich folgende Antikörper näher an:

Anti-alpha Tubulin:

ab15246 (2 Abreviews)

https://www.abcam.com/index.html?datasheet=15246 (or use the following: https://www.abcam.com/index.html?datasheet=15246).

ab18251 (1 Abreview)

https://www.abcam.com/index.html?datasheet=18251 (or use the following: https://www.abcam.com/index.html?datasheet=18251).

Anti-Actin:

ab8227 (5 Abreviews)

https://www.abcam.com/index.html?datasheet=8227 (or use the following: https://www.abcam.com/index.html?datasheet=8227).

Read More

Answer

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 it is one of these genes that have been used as the base sequence for the epitope numbering.

Read More

Answer

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 ab24102.
https://www.abcam.com/index.html?datasheet=29889 https://www.abcam.com/index.html?datasheet=29889.
MRP4 is barely detectable in liver and therefore I recommend to use ab30304 (Prostate (Human) Tissue Lysate - adult normal tissue) as positive control.
https://www.abcam.com/index.html?datasheet=30304 https://www.abcam.com/index.html?datasheet=30304.
With ab63907 we used ab29745 (Placenta (Human) Tissue Lysate - adult normal tissue) successfully as control.
https://www.abcam.com/index.html?datasheet=29745 https://www.abcam.com/index.html?datasheet=29745.
I hope this information is helpful and wish you good luck with your research.

Read More

Question
Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I have issued a free of charge replacement with the order number *****.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Read More

Question
Answer

Thank you for contacting us. I have not been able to find the C. elegans alpha tublin sequence to align with the epitope this clone recognizes (located within the range of chicken alpha tubulin specified on the datasheet). However, I did find a reference that is listed on the datasheet of another version of this clone, DM1A, with the catalogue number ab64503, a FITC-conjugate. Click here (or use the following: https://www.abcam.com/index.html?datasheet=64503). The paper is: Dumont J et al. A kinetochore-independent mechanism drives anaphase chromosome separation during acentrosomal meiosis. Nat Cell Biol 12:894-901 (2010). PubMed: 20729837 The authors used the antibody for immunofluorescent microscopy. Until we receive more data, we will not guarantee ab7291 for reactivity with C. elegans, but I think this paper is encouraging. If you do decide to try it, please consider sending us a review of your resutls. For instructions on how to do this, please see the page at the following link: www.abcam.com/abreviews 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

Question
Answer

Thanks for your call today and for letting us know about the problem with the most recent vial of this antibody. As we discussed, I am sending a vial from a new lot on the order ***, which should arrive promptly. Please keep me updated about how this new vial works, and let me know if there is anything else that we can do for you.

Read More

Answer

Thank you for contacting us. As requested the protocol currently used to stain the HeLa cells with Anti-alpha Tubulin antibody [DM1A] (ab7291) is as follows: The cells were fixed in 100% methanol for 5 minutes and then washed with PBS. The cells were then permeabilised with PBST (0.1%) and blocked for 1 hour in 1% BSA/10% normal serum/0.3M glycine. The block was then washed off using PBST. The primary antibody (5µg/ml) diluted in 1 BSA/PBST was then incubated with the cells overnight at 4 degrees C. The secondary antibody was diluted in the same buffer as the primary antibody and incubated for 1 hour at room temperature. This is not exactly the protocol used to produce the image displayed on the datasheet but one that has been further optimised since and is used fro quality control of the antibody. I have attached an image obtained using the protocol described. I hope this information has been of help. If you are having any difficulty obtaining the staining expected using this antibody or you'd like any further information please do let us know.

Read More

Answer

Thank you for contacting Abcam. I am sorry that the last lot did not perform aswell as previously. I can confirm that we have a different batch of ab7291 in stock and that if you were to order a new vial of the antibody then you would not be getting the same lot as you previously received. If there is anything else I can help with, then please let me know.    

Read More

Answer

Thank you for your enquiry. I'm afraid I was unable to find out this information as I am unable to open publications which have used this clone in electron microscopy, I am very sorry. I enclose below the reference which I hope will have further material and methods details, hopefully you will be able to access this through your institution. Expression and distribution of vanilloid receptor 1 (TRPV1) in the adult rat brain . Attila Tótha, Judit Boczánc, Noémi Kedeia, Erzsébet Lizaneczb, Zsolt Bagib, Zoltán Pappb, István Édesb, László Csibac and Peter M. Blumberga. Molecular Brain Research . Volume 135, Issues 1-2, 27 April 2005, Pages 162-168 http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T07-4F9MTP7-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=c6392ea593b96931bb766ad7040b213c I hope this information helps and I'm sorry I cannot help you more in this occasion.

Read More

Answer

Thank you for your enquiry. The 10 nm filaments are neurofilaments (intracellular protein filaments that are approximately 10 nm in diameter and of indefinite length). To read more about this, please refer to the following reference. Study of the 10-nm-filament fraction isolated during the standard microtubule preparation. Biochem. J. 1980. 191, 543-546 http://www.pubmedcentral.nih.gov/pagerender.fcgi?artid=1162245&pageindex=1 The specificity statement on the datasheet was taken from the following publication. 10-nm Filaments Are Induced to Collapse in Living Cells Microinjected with Monoclonal and Polyclonal Antibodies Against Tubulin. THE JOURNAL OF CELL BIOLOGY VOLUME 9S MARCH 1984 847-858. http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=2113126&blobtype=pdf I hope this information will be helpful. If there is anything else that I can help you with, please do not hesitate to contact me.

Read More

1-10 of 20 Q&A

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com

登録