Anti-alpha Tubulin antibody [DM1A] - Loading Control

• 产品名称

  Anti-alpha Tubulin抗体[DM1A] - Loading Control
  参阅全部 alpha Tubulin 一抗

• 描述

  小鼠单克隆抗体[DM1A] to alpha Tubulin - Loading Control

• 宿主

  Mouse

• 经测试应用

  适用于: ICC/IF, IHC-P, WB, Flow Cyt (Intra)more details

• 种属反应性

  与反应: Mouse, Rat, Human
  预测可用于: Chicken, Guinea pig, Hamster, Cow, Dog, Pig, Xenopus laevis, Gerbil, African green monkey

• 免疫原

  Full length native protein (purified). This information is proprietary to Abcam and/or its suppliers.

• 阳性对照

• WB: HeLa, HEK293, HepG2, Caco2, NIH3T3, PC12 cell lysates. Flow Cyt (Intra): methanol fixed/Tween permeabilised HeLa cells. ICC/IF: Caco-2, NIH3T3, and SV40LT-SMC cells. IHC-P: Human colon and rat colon tissues.

• 常规说明

  This antibody clone [DM1A] is manufactured by Abcam.

  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.

  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.

  
  

  The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

  If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

• 形式

  Liquid

• 存放说明

  Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.

• 存储溶液

  pH: 7.40
  Preservative: 0.02% Sodium azide
  Constituents: PBS, 6.97% L-Arginine

• 浓度

• 100 µg 浓度为 1 mg/ml

• 250 µg 浓度为 1 mg/ml

• 纯度

  Affinity purified

• Primary antibody说明

  Excellent as a protein loading control antibody.

• 克隆

  单克隆

• 克隆编号

  DM1A

• 同种型

  IgG1

• 轻链类型

  kappa

The Abpromise guarantee

Abpromise™承诺保证使用ab7291于以下的经测试应用

“应用说明”部分 下显示的仅为推荐的起始稀释度；实际最佳的稀释度/浓度应由使用者检定。

数据库链接

• Entrez Gene: 7277 Human

• Entrez Gene: 22145 Mouse

• Entrez Gene: 316531 Rat

• Omim: 191110 Human

• SwissProt: P68366 Human

• SwissProt: P68368 Mouse

• SwissProt: Q5XIF6 Rat

• Unigene: 75318 Human

• Unigene: 1155 Mouse

• Unigene: 92961 Rat

别名

• 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

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling alpha Tubulin with ab7291 at a concentration of 0.006ug/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.
  ab7291 anti-alpha Tubulin antibody [DM1A] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.
  Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• 

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

  All lanes : Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution
  
  Lane 1 : HeLa cell lysate
  Lane 2 : PC12 cell lysate
  Lane 3 : SV40LT-SMC cell lysate
  Lane 4 : NIH/3T3 cell lysate
  Lane 5 : Rat liver tissue lysate
  Lane 6 : Rat heart tissue lysate
  
  Lysates/proteins at 20 µg per lane.
  
  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.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)Image from Dai C et al., PLoS One 10(8), Fig 5C. Doi: 10.1371/journal.pone.0063054. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

  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.

• 

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

  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 Alexa Fluor® 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).

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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

• 

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

  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 22°C. 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.

• 

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

  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 Alexa Fluor® 488 (ab150117) at 2 μg/ml (shown in green) and anti-rabbit Alexa Fluor® 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.

• 

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

  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 Alexa Fluor® 488 (ab150117) at 2 μg/ml (shown in green) and anti-rabbit Alexa Fluor® 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.

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)This image is courtesy of an AbReview submitted by Elena Kashuba.

  All lanes : Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/2500 dilution
  
  Lanes 1-2 : Daudi (Human Burkitt's lymphoma cell line) at 10 µg
  Lanes 3-4 : Daudi (Human Burkitt's lymphoma cell line) at 15 µg
  Lanes 5-6 : Daudi (Human Burkitt's lymphoma cell line) at 20 µg
  
  Secondary
  All lanes : 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

• 

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

  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

• 

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

  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.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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.

• 

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

  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).

• 

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

  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.