Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker

14 product images

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  Western blot - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)

  Lanes 1- 2: Merged signal (red and green). Green - ab52623 observed at 52 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 observed at 37 kDa.
  ab52623 Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker was shown to specifically 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 and beta III Tubulin knockout samples were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab52623 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated overnight at 4^°C at 1 in 1000 and 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 [EP1569Y] - Neuronal Marker (ab52623) at 1/1000 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

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  Multiplex immunohistochemistry - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)

  Fluorescence multiplex immunohistochemical analysis of human cerebellum tissue (formalin-fixed paraffin-embedded section).
  

  Merged staining of Neu-N (Anti-NeuN antibody [EPR12763] - Neuronal Marker ab177487; yellow; Opal™570), anti-beta III Tubulin (ab52623; red; Opal™690) and anti-GFAP (Anti-GFAP antibody [EPR1034Y] ab68428; green; Opal™520).

  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ kit.

  The section was incubated in three rounds of staining with Anti-NeuN antibody [EPR12763] - Neuronal Marker ab177487 (1/1000 dilution), ab52623 (1/200 dilution) and Anti-GFAP antibody [EPR1034Y] ab68428 (1/250 dilution); each using a separate fluorescent tyramide signal amplification system.

  Sodium citrate antigen retrieval (pH 6.0) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.

  DAPI (blue) was used as a nuclear counter stain.

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  Immunocytochemistry/ Immunofluorescence - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)

  ab52623 staining beta III Tubulin in wild-type HAP1 cells (top panel) and TUBB3 knockout HAP1 cells (bottom panel).
  

  The cells were fixed with 100% methanol for 5 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated with ab52623 at 1/500 dilution and Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1 hour with a goat secondary antibody to Rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green).

  Nuclear DNA was labeled in blue with DAPI.

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

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  Immunocytochemistry/ Immunofluorescence - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)

  Immunofluorescence staining of beta III Tubulin using ab52623 in ioGlutamatergic Neurons (Human iPSC-Derived Glutamatergic Neurons, ioGlutamatergic Neurons - Human iPSC-Derived Glutamatergic Neurons ab259259), which were differentiated for 11 days post induction.
  

  The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab52623 at 0.1 μg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

  Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

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  Western blot - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)

  Lanes 1- 2: Merged signal (red and green). Green - ab52623 observed at 50 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) observed at 37 kDa.
  

  ab52623 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. ab52623 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) overnight at 4°C at a 1 in 1000 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 [EP1569Y] - Neuronal Marker (ab52623) at 1/1000 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

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  Western blot - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)

  Lanes 1 - 4: Merged signal (red and green). Green - ab52623 observed at 52 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
  

  ab52623 was shown to specifically react with beta III Tubulin in wild-type cells as signal was lost in beta III Tubulin knockout HAP1 cells. Wild-type and beta III Tubulin knockout samples were subjected to SDS-PAGE. ab52623 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623) at 1/1000 dilution

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

  Lane 2: Beta III Tubulin 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

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  Eng et al PLoS One. 2017 May 17;12(5):e0177834. doi: 10.1371/journal.pone.0177834. eCollection 2017. Fig 5. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

  Western blot - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)

  In vivo, CK1δ T347 is phosphorylated in trans.
  

  D) Phosphorylation of CK1δ at T347 is not dependent on CK1 activity. HEK-293 cells over-expressing wild-type CK1δ were treated with CK1 inhibitors PF670462 (PF670, 1 μM), PF4800567 (PF480, 1 μM), or D4476 (30 μM) for 3 hours, followed by addition for 30 min of either DMSO or 40 nM Calyculin A. Cell lysates were immunoblotted for anti-pT347 or anti-Myc antibodies. CK1 inhibitors block the autophosphorylation-induced mobility shift but do not decrease phosphorylation of T347. Myc-tagged CK1δ indicated by curly brackets. ab52623 lower panel.

  All lanes: Western blot - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)

  Predicted band size: 50 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)

  ab52623 staining beta III Tubulin in NGF-differentiated PC-12 (Rat adrenal gland pheochromocytoma cell line) cells.
  

  The cells were fixed with 100% methanol (5 minutes) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1 hour. The cells were then incubated with ab52623 at 5 μg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 at 1 μg/ml overnight at +4°C, followed by a further incubation at room temperature for 1 hour with an AlexaFluor^®488 Goat anti-Rabbit secondary (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and AlexaFluor^®594 Goat anti-Mouse secondary (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labeled in blue with DAPI.
  Negative controls: 1– Rabbit primary 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.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)

  ab52623, at a 1/50 dilution, staining class III beta Tubulin in human breast carcinoma tissue using Immunohistochemistry, Paraffin embedded tissue.
  

  Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

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  Flow Cytometry (Intracellular) - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)

  Intracellular Flow Cytometry analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling beta III Tubulin with ab52623 at 1/20 (red).
  

  Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. An Alexa Fluorr^®488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabeled control, cells without incubation with primary and secondary antibodies.

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  Western blot - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)

  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 ab52623 overnight at 4°C.
  

  Antibody binding was detected using Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) ab175781 at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

  All lanes: Western blot - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623) at 1/1000 dilution

  Lane 1: Brain (Mouse) Tissue Lysate at 20 µg

  Lane 2: Brain (Rat) Tissue Lysate at 20 µg

  Lane 3: Spinal Cord (Mouse) Tissue Lysate at 20 µg

  Lane 4: Spinal Cord (Rat) Tissue Lysate at 20 µg

  Lane 5: PC-12 (Rat adrenal pheochromocytoma cell line) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) ab175781) at 1/10000 dilution

  Predicted band size: 50 kDa

  Observed band size: 52 kDa

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  Flow Cytometry (Intracellular) - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)

  Overlay histogram showing U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) cells stained with ab52623 (red line).
  

  The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 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 (ab52623, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluorr^®488 goat anti-rabbit IgG (H&L) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730, 0.1 μg/1x10⁶) used under the same conditions. Unlabeled sample (blue line) was also used as a control.

  Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

  This antibody gave a positive signal in U-87 MG cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

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  Western blot - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)

  All lanes: Western blot - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623) at 1/10000 dilution

  All lanes: HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

  Secondary

  All lanes: goat anti-rabbit HRP at 1/2000 dilution

  Predicted band size: 50 kDa

  Observed band size: 52 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (ab52623)

  Immunohistochemical analysis of formalin fixed paraffin embedded human breast carcinoma labelling beta III Tubulin with ab52623 at a concentration of 0.5 µg/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, pH 8.5 for 32mins.

  ab52623 anti-beta III Tubulin [EP1569Y] 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).