Anti-beta III Tubulin antibody [EPR19591]

17 product images

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  Immunocytochemistry/ Immunofluorescence - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) cells labeling beta III Tubulin with ab215037 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution (green).
  

  Confocal image showing cytoplasmic staining on U-87 MG cell line.

  The nuclear counterstain is DAPI (blue).

  Tubulin is detected with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/1000 dilution.

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  Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Lanes 1- 4: Merged signal (red and green). Green - ab215037 observed at 52 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) observed at 37 kDa.
  

  ab215037 was shown to react with beta III Tubulin in wild-type HCT116 cells in western blot. Loss of signal was observed when knockout cell line Human TUBB3 (beta III Tubulin) knockout HCT116 cell line ab266900 (knockout cell lysate Human TUBB3 (beta III Tubulin) knockout HCT116 cell lysate ab257070) was used. Wild-type HCT116 and TUBB3 knockout HCT116 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab215037 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037) at 1/2000 dilution

  Lane 1: Wild-type HCT116 cell lysate at 20 µg

  Lane 2: TUBB3 knockout HCT116 cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 50 kDa

  Observed band size: 52 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Immunohistochemical analysis of paraffin-embedded human cholangiocarcinoma tissue labeling beta III Tubulin with ab215037 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.
  

  Cytoplasmic staining on human cholangiocarcinoma is observed.

  Counter stained with Hematoxylin.

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• 

  Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Lanes 1- 2: Merged signal (red and green). Green - ab215037 observed at 50 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) observed at 37 kDa.
  

  ab215037 was shown to react with beta III tubulin in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line Human TUBB3 (beta III Tubulin) knockout HeLa cell line ab255358 (knockout cell lysate Human TUBB3 (beta III Tubulin) knockout HeLa cell lysate ab263857) was used. Wild-type HeLa and TUBB3 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab215037 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037) at 1/2000 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: TUBB3 knockout HeLa cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 50 kDa

  Observed band size: 50 kDa

• 

  Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Lanes 1 - 4: Merged signal (red and green). Green - ab215037 observed at 50 kDa. Red - loading control, Anti-GAPDH antibody [mAbcam 9484] - Loading Control ab9484, observed at 37 kDa.
  

  ab215037 was shown to specifically react with TUBB3 in wild-type HAP1 cells as signal was lost in TUBB3 knockout cells. Wild-type and TUBB3 knockout samples were subjected to SDS-PAGE. ab215037 and Anti-GAPDH antibody [mAbcam 9484] - Loading Control ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1/2000 dilution and 1/20,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037) at 1/2000 dilution

  Lane 1: Wild-type HAP1 whole cell lysate at 20 µg

  Lane 2: TUBB3 knockout HAP1 whole cell lysate at 20 µg

  Lane 3: HeLa whole cell lysate at 20 µg

  Lane 4: HEK293 whole cell lysate at 20 µg

  Predicted band size: 50 kDa

• 

  Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Blocking/Dilution buffer: 5% NFDM/TBST.
  

  Exposure time: Lane 1/7: 1 second; Lane 2/3: 5 seconds; Lane 4/5: 3 minutes; Lane 6:30 seconds.

  Lanes 1 - 5: Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037) at 1/2000 dilution

  Lanes 6 - 7: Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037) at 1/10000 dilution

  Lane 1: Human cerebellum lysate at 20 µg

  Lane 2: U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate at 20 µg

  Lane 3: SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate at 20 µg

  Lane 4: HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

  Lane 5: Human breast cancer lysate at 20 µg

  Lane 6: Human fetal kidney lysate at 10 µg

  Lane 7: Human fetal brain lysate at 10 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

  Predicted band size: 50 kDa

  Observed band size: 50 kDa

• 

  Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Blocking/Dilution buffer: 5% NFDM/TBST.
  

  Exposure time: Lane 1/2: 1 second; Lane 3: 3 minutes; Lane 4/5: 30 seconds.

  Lanes 1 - 2: Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037) at 1/2000 dilution

  Lane 3: Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037) at 1/5000 dilution

  Lanes 4 - 5: Western blot - Anti-beta III Tubulin antibody [EPR19591] (ab215037) at 1/10000 dilution

  Lane 1: Mouse brain lysate at 10 µg

  Lane 2: Rat brain lysate at 10 µg

  Lane 3: Rat heart lysate at 10 µg

  Lane 4: C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

  Lane 5: PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 50 kDa

  Observed band size: 50 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Immunohistochemical analysis of paraffin-embedded human cerebral cortex tissue labeling beta III Tubulin with ab215037 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.
  

  Cytoplasmic staining on human cerebral cortex is observed.

  Counter stained with Hematoxylin.

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Immunohistochemical analysis of paraffin-embedded human glioma tissue labeling beta III Tubulin with ab215037 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.
  

  Cytoplasmic staining on human glioma is observed.

  Counter stained with Hematoxylin.

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma tissue labeling beta III Tubulin with ab215037 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.
  

  Negative staining on human hepatocellular carcinoma. (PMID: 25039376).

  Counter stained with Hematoxylin.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• 

  Immunoprecipitation - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  beta III Tubulin was immunoprecipitated from 0.35 mg of U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate with ab215037 at 1/30 dilution.
  

  Western blot was performed from the immunoprecipitate using ab215037 at 1/1000 dilution.

  VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/10,000 dilution

  Lane 1: U-87 MG whole cell lysate 10μg (Input).

  Lane 2: ab215037 IP in U-87 MG whole cell lysate.

  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab215037 in U-87 MG whole cell lysate.

  Blocking and dilution buffer and concentration: 5% NFDM/TBST.

  Exposure time: 10 seconds.

  All lanes: Immunoprecipitation - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Predicted band size: 50 kDa

  Observed band size: 50 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma tissue labeling beta III Tubulin with ab215037 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.
  

  Cytoplasmic staining on human lung adenocarcinoma is observed.

  Counter stained with Hematoxylin.

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Immunohistochemical analysis of paraffin-embedded mouse cerebral cortex tissue labeling beta III Tubulin with ab215037 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.
  

  Cytoplasmic staining on mouse cerebral cortex is observed.

  Counter stained with Hematoxylin.

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Immunohistochemical analysis of paraffin-embedded rat cerebral cortex tissue labeling beta III Tubulin with ab215037 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.
  

  Cytoplasmic staining on rat cerebral cortex is observed.

  Counter stained with Hematoxylin.

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SH-SY5Y (Human neuroblastoma cell line from bone marrow) cells labeling beta III Tubulin with ab215037 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution (green).
  

  Confocal image showing cytoplasmic staining on SH-SY5Y cell line.

  The nuclear counterstain is DAPI (blue).

  Tubulin is detected with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/1000 dilution.

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  Flow Cytometry (Intracellular) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) cells labeling beta III Tubulin with ab215037 at 1/200 dilution (red) compared with a rabbit monoclonal IgG isotype control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.
  

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  Flow Cytometry (Intracellular) - Anti-beta III Tubulin antibody [EPR19591] (ab215037)

  Flow cytometry overlay histogram showing wild-type Hap1 (green line) and TUB3 knockout Hap1 stained with ab215037 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab215037) (1x 106 in 100μl at 0.2 μg/ml (1/10400)) for 30min at 22°C.
  
  The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C
  
  Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type Hap1 - black line, TUB3 knockout Hap1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).
  
  Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.
  
  This antibody gave a positive signal in Hap1 Fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.