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

• ICC/IF

Lab

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, 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 ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and 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.

• IHC-P

Lab

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).

• WB

Lab

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 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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-alexa-fluor-790-ab175781'>ab175781</a>) at 1/10000 dilution

Predicted band size: 50 kDa

Observed band size: 52 kDa

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• Flow Cyt (Intra)

Unknown

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) (ab150081) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (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.

• ICC/IF

Unknown

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 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 (ab150081) at 2 μg/ml (shown in green) and AlexaFluor^®594 Goat anti-Mouse secondary (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.

• Flow Cyt (Intra)

Unknown

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.

• ICC/IF

Unknown

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 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) (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).

• IHC-P

Unknown

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.

• mIHC

Lab

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 (ab177487; yellow; Opal™570), anti-beta III Tubulin (ab52623; red; Opal™690) and anti-GFAP (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 ab177487 (1/1000 dilution), ab52623 (1/200 dilution) and 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.

• WB

Lab

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 (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 ab255358 (knockout 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 (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 (ab216773) and 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

Lane 2:

Western blot - Human TUBB3 (beta III Tubulin) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-tubb3-beta-iii-tubulin-knockout-hela-cell-line-ab255358'>ab255358</a>)

Predicted band size: 50 kDa

Observed band size: 50 kDa

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• WB

Unknown

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, 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 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 ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed 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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• WB

Unknown

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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• WB

Lab

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 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 ab266900 (knockout 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 (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 (ab216773) and 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

Lane 2:

Western blot - Human TUBB3 (beta III Tubulin) knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-tubb3-beta-iii-tubulin-knockout-hct116-cell-line-ab266900'>ab266900</a>)

Predicted band size: 50 kDa

Observed band size: 52 kDa

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• WB

PubMed

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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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/

• WB

CiteAb

Western blot - Anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker (AB52623)

beta III Tubulin western blot using anti-beta III Tubulin antibody [EP1569Y] - Neuronal Marker ab52623. Publication image and figure legend from Zhang, H., Qi, Y., et al., 2017, Sci Rep, PubMed 28165507.

ab52623 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab52623 please see the product overview.

PF inhibited IL-6 production of BMDCs and impaired Th17 cell differentiation via suppressing IKK/NF-κB and MAPK signaling pathway in vitro.Bone marrow cells from naïve mice were stimulated by GM-CSF and IL-4 to induce DC differentiation in the presence or absence of the indicated concentrations of PF for 5 days, and followed by the stimulation of LPS. (A) The above DCs were co-cultured with naïve CD4+ T cells for Th17 cell differentiation. The percentage of Th17 cells were analyzed by flow cytometry. (B) The protein expression of IKK/NF-κB signaling pathway was analyzed by immunoblot assay. Tubulin served as control. (C) The nucleus translocation of NF-κB-p65 was monitored by fluorescence microscopy (400×). Nuclei were stained in blue; the NF-κB p65 was stained in red. (D) Protein expression of MAPK signaling pathway was detected by immunoblot assay. β-actin was detected as control. Data are representative of three independent experiments. Values were expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

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