Anti-alpha Tubulin antibody - Loading Control

• WB

Lab

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB18251)

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 5% Milk before being incubated with ab18251 overnight at 4°C. Antibody binding was detected using ab175772 at a 1 : 10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

All lanes:

Western blot - Anti-alpha Tubulin antibody - Loading Control (ab18251) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 5 µg

Lane 3:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 2 µg

Lane 4:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 1 µg

Secondary

All lanes:

Western blot - Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 680) (<a href='/en-us/products/secondary-antibodies/donkey-rabbit-igg-h-l-alexa-fluor-680-ab175772'>ab175772</a>) at 1/10000 dilution

Predicted band size: 36 kDa

Observed band size: 52 kDa

false

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-alpha Tubulin antibody - Loading Control (AB18251)

Overlay histogram showing Caco2 cells stained with ab18251 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween 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 (ab18251, 0.01μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/4000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (polyclonal) (ab27478, 0.01μg/1x10⁶ cells) 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody - Loading Control (AB18251)

ab18251 staining alpha-Tubulin in Caco-2 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 ab18251 at 5 μl/ml and ab7291 at 1 μg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-rabbit Alexa Fluor^® 488 (ab150081) at 2 μg/ml (shown in green) and anti-mouse Alexa Fluor^® 594 (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

Negative controls : 1– Rabbit primary antibody 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.

• ICC/IF

AbReview4208****

Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody - Loading Control (AB18251)

ab18251 at a 1/8000 dilution staining human HeLa cells by immunocytochemistry. The cells were paraformaldehyde fixed and incubated with the antibody for 30 minutes. The secondary antibody was a Cy3^® conjugated Goat Anti-Rabbit IgG (H+L). The image shows staining of an interphase IM cell.

This image is courtesy of an Abreview by Kirk McManus submitted on 27 February 2006.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody - Loading Control (AB18251)

ab18251 staining alpha Tubulin in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-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 overnight at 4°C with ab18251 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 100% methanol (5 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-alpha Tubulin antibody - Loading Control (AB18251)

Overlay histogram showing NIH3T3 cells stained with ab18251 (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 (ab18251, 0.01μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/4000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (polyclonal) (ab27478, 0.01μg/1x10⁶ cells) 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody - Loading Control (AB18251)

ab18251 staining alpha-Tubulin in NIH3T3 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 ab18251 at 1 μl/ml and ab7291 at 1μg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-rabbit Alexa Fluor^® 488 (ab150081) at 2 μg/ml (shown in green) and anti-mouse Alexa Fluor^® 594 (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

Negative controls : 1– Rabbit primary antibody 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.

• WB

Lab

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB18251)

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 5% Milk before being incubated with ab18251 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-alpha Tubulin antibody - Loading Control (ab18251) at 1 µg/mL

All lanes:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (ab27252) at 10 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: 36 kDa

Observed band size: 50 kDa

false

• WB

Lab

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB18251)

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 5% Milk before being incubated with ab18251 overnight at 4°C. Antibody binding was detected using ab175780 at a 1 : 10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

All lanes:

Western blot - Anti-alpha Tubulin antibody - Loading Control (ab18251) at 1 µg/mL

All lanes:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 790) (<a href='/en-us/products/secondary-antibodies/donkey-rabbit-igg-h-l-alexa-fluor-790-ab175780'>ab175780</a>) at 1/10000 dilution

Predicted band size: 36 kDa

Observed band size: 50 kDa

false

• WB

Lab

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB18251)

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 5% Milk before being incubated with ab18251 overnight at 4°C. Antibody binding was detected using ab175773 at a 1 : 10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

All lanes:

Western blot - Anti-alpha Tubulin antibody - Loading Control (ab18251) at 1 µg/mL

All lanes:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 680) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-alexa-fluor-680-ab175773'>ab175773</a>) at 1/10000 dilution

Predicted band size: 36 kDa

Observed band size: 50 kDa

false

• WB

Lab

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB18251)

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 2% Bovine Serum Albumin before being incubated with ab18251 overnight at 4°C. Antibody binding was detected using an anti-rabbit HRP (ab97051), and visualised using ECL development solution ab133406

All lanes:

Western blot - Anti-alpha Tubulin antibody - Loading Control (ab18251) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

A431 (Human epithelial carcinoma 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 1/50000 dilution

Predicted band size: 50 kDa

Observed band size: 50 kDa

true

Exposure time: 1min

• WB

Lab

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB18251)

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 ab18251 overnight at 4°C. Antibody binding was detected using Anti-Rabbit 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-alpha Tubulin antibody - Loading Control (ab18251) at 0.5 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

HEK-293 (Human embryonic kidney cell line) whole cell lysate at 20 µg

Lane 3:

HepG2 (Human hepatocellular liver carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

Caco-2 (Human colonic carcinoma cell line) whole cell lysate at 20 µg

Lane 5:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 20 µg

Lane 6:

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: 27 kDa,50 kDa

Observed band size: 44 kDa,52 kDa

false

• WB

Project

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB18251)

ab18251 detects a strong band at 50 kDa corresponding to alpha tubulin. Cross-reactivity is also seen with other lower molecular weight bands. This may be reduced by using the antibody at a lower working concentration.

All lanes:

Western blot - Anti-alpha Tubulin antibody - Loading Control (ab18251) at 0.5 µg/mL

Lane 1:

HeLa lysate at 20 µg

Lane 2:

A431 lysate at 20 µg

Secondary

All lanes:

Alexa Flour Goat polyclonal to Rabbit IgG (700) at 1/10000 dilution

Predicted band size: 50 kDa

Observed band size: 30 kDa,50 kDa

false

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody - Loading Control (AB18251)

ab18251 staining alpha-Tubulin in SV40LT-SMC cells.

The cells were fixed with 100% methanol for 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 ab18251 at 1 μl/ml and ab7291 at 1 μg/ml overnight at +4°C, followed by a further incubation at room temperature for 1 hour with an anti-rabbit Alexa Fluor^® 488 (ab150081) at 2 μg/ml (shown in green) and anti-mouse Alexa Fluor^® 594 (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labeled in blue with DAPI.

Negative controls : 1– Rabbit primary antibody 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)

Lab

Flow Cytometry (Intracellular) - Anti-alpha Tubulin antibody - Loading Control (AB18251)

Overlay histogram showing SV40LT-SMC cells stained with ab18251 (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 (ab18251, 0.01μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/4000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (polyclonal) (ab27478, 0.01μg/1x10⁶ cells) 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.