Anti-beta III Tubulin antibody [EPR19591] - Neuronal Marker

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-beta III Tubulin antibody [EPR19591] - Neuronal Marker (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.

• IHC-P

Supplier Data

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

• IHC-P

Supplier Data

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

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-beta III Tubulin antibody [EPR19591] - Neuronal Marker (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 (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.

• IHC-P

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-beta III Tubulin antibody [EPR19591] - Neuronal Marker (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) (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 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) (ab150077) at 1/1000 dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-beta III Tubulin antibody [EPR19591] - Neuronal Marker (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) (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 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) (ab150077) at 1/1000 dilution.

• IHC-P

Supplier Data

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

• IHC-P

Supplier Data

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

• IP

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

Predicted band size: 50 kDa

Observed band size: 50 kDa

false

• IHC-P

Supplier Data

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

• IHC-P

Supplier Data

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

• WB

Lab

Western blot - Anti-beta III Tubulin antibody [EPR19591] - Neuronal Marker (AB215037)

Lanes 1- 4 : Merged signal (red and green). Green - ab215037 observed at 52 kDa. Red - 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 ab266900 (knockout 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 (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 (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 [EPR19591] - Neuronal Marker (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

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

false

• WB

Lab

Western blot - Anti-beta III Tubulin antibody [EPR19591] - Neuronal Marker (AB215037)

Lanes 1- 2 : Merged signal (red and green). Green - ab215037 observed at 50 kDa. Red - 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 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. ab215037 and 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 (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 [EPR19591] - Neuronal Marker (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

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

false

• WB

Lab

Western blot - Anti-beta III Tubulin antibody [EPR19591] - Neuronal Marker (AB215037)

Lanes 1 - 4 : Merged signal (red and green). Green - ab215037 observed at 50 kDa. Red - 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 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (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] - Neuronal Marker (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

false

• WB

Supplier Data

Western blot - Anti-beta III Tubulin antibody [EPR19591] - Neuronal Marker (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] - Neuronal Marker (ab215037) at 1/2000 dilution

Lanes 6 - 7:

Western blot - Anti-beta III Tubulin antibody [EPR19591] - Neuronal Marker (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

false

• WB

Supplier Data

Western blot - Anti-beta III Tubulin antibody [EPR19591] - Neuronal Marker (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] - Neuronal Marker (ab215037) at 1/2000 dilution

Lane 3:

Western blot - Anti-beta III Tubulin antibody [EPR19591] - Neuronal Marker (ab215037) at 1/5000 dilution

Lanes 4 - 5:

Western blot - Anti-beta III Tubulin antibody [EPR19591] - Neuronal Marker (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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 50 kDa

Observed band size: 50 kDa

false