Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (AB221935)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (AB221935)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labelling beta III Tubulin with ab52623 at 1/20 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. An Alexa Fluor^®488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52623).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (AB221935)

This ICC/IF data was generated using the same anti-beta III tubulin antibody clone, EP1569Y, in a different buffer formulation (cat# 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 (5min), 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 1h. 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 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (AB221935)

ab52623 staining beta III Tubulin in NGF-differentiated PC12 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 ab52623 at 5μg/ml and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h 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 labelled 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52623).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (AB221935)

Overlay histogram showing U-87MG 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 Fluor^® 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, 0.1μg/1x10⁶) used under the same conditions. Unlabelled 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-87MG cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52623).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (AB221935)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52623).

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

• WB

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Western blot - Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (AB221935)

This WB data was generated using the same anti-beta III tubulin antibody clone, EP1569Y, in a different buffer formulation (cat# 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 (<a href='/en-us/products/primary-antibodies/beta-iii-tubulin-antibody-ep1569y-neuronal-marker-ab52623'>ab52623</a>) 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

false

• WB

Lab

Western blot - Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (AB221935)

This data was developed using the same antibody clone in a different buffer formulation (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 (<a href='/en-us/products/primary-antibodies/beta-iii-tubulin-antibody-ep1569y-neuronal-marker-ab52623'>ab52623</a>) 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

false

• WB

Lab

Western blot - Anti-beta III Tubulin antibody [EP1569Y] - BSA and Azide free (AB221935)

This data was developed using the same antibody clone in a different buffer formulation (ab52623).

Lanes 1- 4 : Merged signal (red and green). Green - ab52623 observed at 52 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 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. 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 (<a href='/en-us/products/primary-antibodies/beta-iii-tubulin-antibody-ep1569y-neuronal-marker-ab52623'>ab52623</a>) 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

false