Anti-alpha Tubulin antibody [DM1A] - Loading Control

41 product images

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  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Merged signal (red and green). Green - ab7291 observed at 52 kDa. Red - loading control, Anti-GAPDH antibody [EPR16891] - Loading Control ab181602, observed at 38 kDa.
  

  All samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. ab7291 and Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1/1,000 and 1/20,000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution

  Lane 1: HeLa cell lysate at 20 µg

  Lane 2: PC12 cell lysate at 20 µg

  Lane 3: SV40LT-SMC cell lysate at 20 µg

  Lane 4: NIH/3T3 cell lysate at 20 µg

  Lane 5: Rat liver tissue lysate at 20 µg

  Lane 6: Rat heart tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 50 kDa

• 

  This image is courtesy of a customer review submitted by Elena Kashuba.

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  All lanes: Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/2500 dilution

  Lanes 1 - 2: Daudi (Human Burkitt's lymphoma cell line) at 10 µg

  Lanes 3 - 4: Daudi (Human Burkitt's lymphoma cell line) at 15 µg

  Lanes 5 - 6: Daudi (Human Burkitt's lymphoma cell line) at 20 µg

  Secondary

  All lanes: HRP conjugated monoclonal Goat Anti-Mouse IgG at 1/1000 dilution

  Performed under reducing conditions.

  Predicted band size: 50 kDa

  Observed band size: 50 kDa

  Exposure time: 1min

• 

  Flow Cytometry (Intracellular) - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Overlay histogram showing HeLa cells stained with ab7291 (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 (ab7291, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was an anti-mouse DyLight^® 488 (Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (Mouse IgG1, Kappa Monoclonal [B11/6] - Isotype Control ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Goat Anti-Mouse IgG H&L (Alexa Fluor® 790) preadsorbed ab186695 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 [DM1A] - Loading Control (ab7291) 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-Mouse IgG H&L (Alexa Fluor® 790) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 790) preadsorbed ab186695) at 1/10000 dilution

  Predicted band size: 36 kDa

  Observed band size: 50 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Alpha Tubulin Western blot staining using mouse Anti-alpha Tubulin antibody

  Lanes 1-3: Merged signal (red and green). Green - Anti-ketohexokinase antibody [EPR15847] ab197593 observed at 33 kDa. Red - loading control ab7291 observed at 50 kDa.

  Anti-ketohexokinase antibody [EPR15847] ab197593 Anti-ketohexokinase antibody [EPR15847] was shown to specifically react with ketohexokinase in wild-type HEK293T cells. Loss of signal was observed when knockout cell line Human KHK (ketohexokinase) knockout HEK-293T cell line ab267292 (knockout cell lysate Human KHK (ketohexokinase) knockout HEK-293T cell lysate ab258017) was used. Wild-type and ketohexokinase knockout samples were subjected to SDS-PAGE. Anti-ketohexokinase antibody [EPR15847] ab197593 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 dilution 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-ketohexokinase antibody [EPR15847] (Anti-ketohexokinase antibody [EPR15847] ab197593) at 1/1000 dilution

  Lane 1: Wild-type HEK293T cell lysate at 20 µg

  Lane 2: KHK knockout HEK293T cell lysate at 20 µg

  Lane 3: Human kidney tissue lysate at 20 µg

  Secondary

  Lanes 1 - 3: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 3: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Predicted band size: 33 kDa

  Observed band size: 33 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Goat Anti-Mouse IgG H&L (Alexa Fluor® 750) ab175740 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 [DM1A] - Loading Control (ab7291) 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-Mouse IgG H&L (Alexa Fluor® 750) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 750) ab175740) at 1/10000 dilution

  Predicted band size: 36 kDa

  Observed band size: 50 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Lanes 1 - 2: Merged signal (red and green). Green - ADAM17 observed at 120 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa. Rabbit anti-ADAM17 antibody was shown to react with TACE in wild-type cells in Western blot with loss of signal observed in ADAM17 knockout cell line Human ADAM17 knockout HCT116 cell line ab266873 (ADAM17 knockout cell lysate Human ADAM17 knockout HCT116 cell lysate ab256832). Wild-type and ADAM17 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with rabbit anti-ADAM17 antibody and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

  All lanes: Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution

  Lane 1: Wild-type cell lysate

  Lane 2: Western blot - Human ADAM17 knockout HCT116 cell line (Human ADAM17 knockout HCT116 cell line ab266873)

  Secondary

  Lane 1: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lane 2: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 93 kDa

  Observed band size: 120 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Goat Anti-Mouse IgG H&L (Alexa Fluor® 750) preadsorbed ab175741 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 [DM1A] - Loading Control (ab7291) 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-Mouse IgG H&L (Alexa Fluor® 750) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 750) preadsorbed ab175741) at 1/10000 dilution

  Predicted band size: 36 kDa

  Observed band size: 50 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Goat Anti-Mouse IgG H&L (Alexa Fluor® 680) preadsorbed ab186694 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 [DM1A] - Loading Control (ab7291) 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-Mouse IgG H&L (Alexa Fluor® 680) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 680) preadsorbed ab186694) at 1/10000 dilution

  Predicted band size: 36 kDa

  Observed band size: 50 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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/0.1% TBST blocking buffer before being incubated with ab7291 overnight at 4°C. Antibody binding was detected using Goat Anti-Armenian hamster IgG H&L (Alexa Fluor® 750) ab175760 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 [DM1A] - Loading Control (ab7291) at 1 µg/mL

  All lanes: HeLa Whole Cell Lysate at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Armenian hamster IgG H&L (Alexa Fluor® 750) (Goat Anti-Armenian hamster IgG H&L (Alexa Fluor® 750) ab175760) at 1/10000 dilution

  Performed under reducing conditions.

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Donkey Anti-Mouse IgG H&L (Alexa Fluor® 750) preadsorbed ab175739 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 [DM1A] - Loading Control (ab7291) 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-Mouse IgG H&L (Alexa Fluor® 750) preadsorbed (Donkey Anti-Mouse IgG H&L (Alexa Fluor® 750) preadsorbed ab175739) at 1/10000 dilution

  Predicted band size: 36 kDa

  Observed band size: 50 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Alpha Tubulin Western blot staining using mouse Anti-alpha Tubulin antibody

  Lane 1: Wild-type HeLa cell lysate 20 μg
  Lane 2: HIST1H1C knockout HeLa cell lysate 20 μg
  False colour image of Western blot: Anti-Histone H1.2 antibody staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-Histone H1.2 antibody ab17677 was shown to bind specifically to Histone H1.2. A band was observed at 32 kDa in wild-type HeLa cell lysates with no signal observed at this size in HIST1H1C knockout cell line Human HIST1H1C (Histone H1.2) knockout HeLa cell line ab261794 (knockout cell lysate Human HIST1H1C (Histone H1.2) knockout HeLa cell lysate ab257218). To generate this image, wild-type and HIST1H1C knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were 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) at 1/20000 dilution.

  All lanes: Western blot - Anti-Histone H1.2 antibody (Anti-Histone H1.2 antibody ab17677) at 1/1000 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: HIST1H1C knockout HeLa cell lysate at 20 µg

  Secondary

  Lanes 1 - 2: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 2: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 21 kDa

  Observed band size: 32 kDa, 50 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Donkey F(ab')2 Anti-Mouse IgG H&L (Alexa Fluor® 750) ab175737 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 [DM1A] - Loading Control (ab7291) at 1 µg/mL

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

  Secondary

  All lanes: Western blot - Donkey F(ab')2 Anti-Mouse IgG H&L (Alexa Fluor® 750) (Donkey F(ab')2 Anti-Mouse IgG H&L (Alexa Fluor® 750) ab175737) at 1/10000 dilution

  Predicted band size: 36 kDa

  Observed band size: 50 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Alpha Tubulin Western blot staining using mouse Anti-alpha Tubulin antibody

  Lanes 1 - 4: Merged signal (red and green). Green - Anti-DDAH1 antibody [EPR13922] ab180599 observed at 37 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.
  

  Anti-DDAH1 antibody [EPR13922] ab180599 was shown to react with DDAH1 in A431 wild-type cells in Western blot. Loss of signal was observed when DDAH1 knockout sample was used. A431 wild-type and DDAH1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween®) before incubation with Anti-DDAH1 antibody [EPR13922] ab180599 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 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 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-DDAH1 antibody [EPR13922] (Anti-DDAH1 antibody [EPR13922] ab180599) at 1/10000 dilution

  Lane 1: Wild-type A431 cell lysate at 40 µg

  Lane 2: DDAH1 knockout A431 cell lysate at 40 µg

  Lane 3: HUVEC (Human umbilical vein endothelial cell line) whole cell lysate at 40 µg

  Lane 4: HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 40 µg

  Secondary

  Lanes 1 - 4: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 4: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 31 kDa

  Observed band size: 37 kDa, 55 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Lanes 1 - 2: Merged signal (red and green). Green - ADAM17 observed at 120 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa. Rabbit anti-ADAM17 antibody was shown to react with TACE in wild-type cells in Western blot with loss of signal observed in ADAM17 knockout cell line Human ADAM17 knockout HeLa cell line ab264911 (ADAM17 knockout cell lysate Human ADAM17 knockout HeLa cell lysate ab256829). Wild-type and ADAM17 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with rabbit anti-ADAM17 antibody and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

  Lane 1: Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution

  Lane 2: Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/20000 dilution

  Lane 1: Western blot - Human ADAM17 knockout HeLa cell line (Human ADAM17 knockout HeLa cell line ab264911)

  Lane 2: Western blot - Human ADAM17 knockout HeLa cell lysate (Human ADAM17 knockout HeLa cell lysate ab256829)

  Secondary

  All lanes: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/2000 dilution

  Performed under reducing conditions.

  Predicted band size: 93 kDa

  Observed band size: 120 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Donkey Anti-Mouse IgG H&L (Alexa Fluor® 680) ab175774 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 [DM1A] - Loading Control (ab7291) 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-Mouse IgG H&L (Alexa Fluor® 680) (Donkey Anti-Mouse IgG H&L (Alexa Fluor® 680) ab175774) at 1/10000 dilution

  Predicted band size: 36 kDa

  Observed band size: 52 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Goat Anti-Mouse IgG H&L (Alexa Fluor® 680) ab175775 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 [DM1A] - Loading Control (ab7291) 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-Mouse IgG H&L (Alexa Fluor® 680) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 680) ab175775) at 1/10000 dilution

  Predicted band size: 36 kDa

  Observed band size: 50 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Donkey Anti-Mouse IgG H&L (Alexa Fluor® 680) preadsorbed ab186698 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 [DM1A] - Loading Control (ab7291) 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-Mouse IgG H&L (Alexa Fluor® 680) preadsorbed (Donkey Anti-Mouse IgG H&L (Alexa Fluor® 680) preadsorbed ab186698) at 1/10000 dilution

  Predicted band size: 36 kDa

  Observed band size: 50 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Donkey Anti-Mouse IgG H&L (Alexa Fluor® 790) preadsorbed ab186699 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 [DM1A] - Loading Control (ab7291) 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-Mouse IgG H&L (Alexa Fluor® 790) preadsorbed (Donkey Anti-Mouse IgG H&L (Alexa Fluor® 790) preadsorbed ab186699) at 1/10000 dilution

  Predicted band size: 36 kDa

  Observed band size: 50 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 blocking buffer before being incubated with ab7291 overnight at 4°C. Antibody binding was detected using Rabbit Anti-Sheep IgG H&L (Alexa Fluor® 750) ab175757 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 [DM1A] - Loading Control (ab7291) at 1 µg/mL

  All lanes: HeLa Whole Cell Lysate at 10 µg

  Secondary

  All lanes: Western blot - Rabbit Anti-Sheep IgG H&L (Alexa Fluor® 750) (Rabbit Anti-Sheep IgG H&L (Alexa Fluor® 750) ab175757) at 1/10000 dilution

  Performed under reducing conditions.

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using an anti-mouse HRP (Goat Anti-Mouse IgG H&L (HRP) preadsorbed ab97040), and visualised using ECL development solution ECL Substrate Kit (High Sensitivity) ab133406.

  All lanes: Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1 µg/mL

  Lane 1: HeLa (Human epithelial cell line from cervix adenocarcinoma cell line) whole cell lysate at 10 µg

  Lane 2: NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

  Lane 3: PC12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (Goat Anti-Mouse IgG H&L (HRP) preadsorbed ab97040) at 1/50000 dilution

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 36 kDa, 50 kDa

  Observed band size: 50 kDa

  Exposure time: 150s

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Donkey Anti-Mouse IgG H&L (Alexa Fluor® 790) ab175782 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 [DM1A] - Loading Control (ab7291) 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-Mouse IgG H&L (Alexa Fluor® 790) (Donkey Anti-Mouse IgG H&L (Alexa Fluor® 790) ab175782) at 1/10000 dilution

  Predicted band size: 36 kDa

  Observed band size: 50 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Goat Anti-Mouse IgG H&L (HRP) ab205719, and visualised using ECL development solution ECL Substrate Kit (High Sensitivity) ab133406.

  All lanes: Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1 µg/mL

  Lane 1: Liver (Human) Tissue Lysate at 10 µg

  Lane 2: Liver (Mouse) Tissue Lysate at 10 µg

  Lane 3: Liver (Rat) Tissue Lysate at 10 µg

  Lane 4: HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

  Lane 5: NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

  Lane 6: PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Mouse IgG H&L (HRP) (Goat Anti-Mouse IgG H&L (HRP) ab205719) at 1/5000 dilution

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 36 kDa

  Observed band size: 52 kDa

  Exposure time: 5s

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Donkey Anti-Mouse IgG H&L (Alexa Fluor® 750) ab175738 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 [DM1A] - Loading Control (ab7291) 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-Mouse IgG H&L (Alexa Fluor® 750) (Donkey Anti-Mouse IgG H&L (Alexa Fluor® 750) ab175738) at 1/10000 dilution

  Predicted band size: 33 kDa, 36 kDa, 42 kDa, 47 kDa, 61 kDa, 80 kDa

  Observed band size: 100 kDa, 50 kDa, 60 kDa, 74 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  ab7291 staining alpha Tubulin in SV40LT-SMC cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes 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 ab7291 at a working concentration of 0.5μg/ml and Alexa Fluor® 647 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker ab190573, Rabbit monoclonal [EP1332Y] to alpha Tubulin (Alexa Fluor^® 647, shown in red) at 1/250 overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-mouse Alexa Fluor^® 488 (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
  

  This product also gave a positive signal in 100% methanol (5 min) fixed SV40 cells under the same testing conditions.

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

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Goat Anti-Mouse IgG H&L (HRP) ab205719 (Left Image) and a competitor secondary (Right Image), and visualised using ECL development solution ECL Substrate Kit (High Sensitivity) ab133406.

  All lanes: Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1 µg/mL

  Lane 1: Liver (Human) Tissue Lysate at 10 µg

  Lane 2: Liver (Mouse) Tissue Lysate at 10 µg

  Lane 3: Liver (Rat) Tissue Lysate at 10 µg

  Secondary

  All lanes: Goat Anti-Mouse IgG H&L (HRP) ab205719 (Left Image) at 1/5000 and a competitor secondary (Right Image) at 1/5000. Notice the decreased signal of the competitor product.

  Performed under reducing conditions.

  Predicted band size: 36 kDa

  Observed band size: 52 kDa

  Exposure time: 5s

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Western blot: Anti-beta 2 Microglobulin antibody [EP2978Y] staining at 1/5000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A]  (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-beta 2 Microglobulin antibody [EP2978Y] ab75853 was shown to bind specifically to beta 2 Microglobulin. A band was observed at 12 kDa in Wild-type A431 and HEK-293T cell lysates with no signal observed at this size in B2M (beta 2 Microglobulin) knockout A-431 cell line Human B2M (beta 2 Microglobulin) knockout A-431 cell line ab261893 (knockout cell lysate Human B2M (beta 2 Microglobulin) knockout A-431 cell lysate ab261702) or B2M knockout HEK-293T cell line Human B2M (beta 2 Microglobulin) knockout HEK-293T cell line ab266828 (knockout cell lysate Human B2M (beta 2 Microglobulin) knockout HEK-293T cell lysate ab256845). To generate this image, wild-type and B2M knockout A431 and HEK-293T cell lysates were analysed.
  Nitrocellulose membranes were blocked in fluorescent western blot (TBS-based) blocking solution in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

  All lanes: Western blot - Anti-beta 2 Microglobulin antibody [EP2978Y] (Anti-beta 2 Microglobulin antibody [EP2978Y] ab75853) at 1/5000 dilution

  Lane 1: Wild-type HEK-293T cell lysate at 20 µg

  Lane 2: B2M knockout HEK-293T cell lysate at 20 µg

  Lane 2: Western blot - Human B2M (beta 2 Microglobulin) knockout HEK-293T cell line (Human B2M (beta 2 Microglobulin) knockout HEK-293T cell line ab266828)

  Lane 2: Western blot - Human B2M (beta 2 Microglobulin) knockout HEK-293T cell lysate (Human B2M (beta 2 Microglobulin) knockout HEK-293T cell lysate ab256845)

  Lane 3: Wild-type A431 cell lysate at 20 µg

  Lane 4: B2M knockout A431 cell lysate at 20 µg

  Lane 4: Western blot - Human B2M (beta 2 Microglobulin) knockout A-431 cell line (Human B2M (beta 2 Microglobulin) knockout A-431 cell line ab261893)

  Lane 4: Western blot - Human B2M (beta 2 Microglobulin) knockout A-431 cell lysate (Human B2M (beta 2 Microglobulin) knockout A-431 cell lysate ab261702)

  Performed under reducing conditions.

  Predicted band size: 14 kDa

  Observed band size: 12 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  ab7291 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 ab7291 at 1μl/ml and Anti-beta Tubulin antibody - Loading Control ab6046 at 1μg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-mouse Alexa Fluor^® 488 (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) at 2 μg/ml (shown in green) and anti-rabbit Alexa Fluor^® 594 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed ab150088) 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.

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Anti-Mouse Alexa Fluor® 790 (Goat Anti-Mouse IgG H&L (Alexa Fluor® 790) ab175783) 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 [DM1A] - Loading Control (ab7291) at 1/5000 dilution

  Lane 1: HeLa (Human epithelial cell line from cervix adenocarcinoma) Whole Cell Lysate at 20 µg

  Lane 2: HEK293 (Human epithelial cell line from embryonic kidney) Whole Cell Lysate at 20 µg

  Lane 3: HepG2 (Human liver hepatocellular carcinoma cell line) Whole Cell Lysate at 20 µg

  Lane 4: Caco 2 (Human colorectal adenocarcinoma 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: PC12 (Rat adrenal gland pheochromocytoma cell line) Whole Cell Lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Mouse IgG H&L (Alexa Fluor® 790) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 790) ab175783) at 1/10000 dilution

  Predicted band size: 36 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  IHC image of ab7291 staining alpha Tubulin in human colon formalin fixed paraffin embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab7291, 5ug/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
  

  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• 

  Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  ab7291 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 ab7291 at 1μg/ml and Anti-beta Tubulin antibody - Loading Control ab6046 at 1μg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-mouse Alexa Fluor^® 488 (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) at 2 μg/ml (shown in green) and anti-rabbit Alexa Fluor^® 594 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed ab150088) 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.

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  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 ab7291 overnight at 4°C. Antibody binding was detected using Goat Anti-Mouse IgG H&L (Alexa Fluor® 790) ab175783 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 [DM1A] - Loading Control (ab7291) 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-Mouse IgG H&L (Alexa Fluor® 790) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 790) ab175783) at 1/10000 dilution

  Predicted band size: 36 kDa

  Observed band size: 50 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Immunofluorescence staining of GABA Transporter 1 / GAT 1 using Anti-GABA Transporter 1 / GAT 1 antibody [EPR24202-20] ab259971 in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. 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 Anti-GABA Transporter 1 / GAT 1 antibody [EPR24202-20] ab259971 at 1 μg/ml and 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.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  IHC image of ab7291 staining alpha Tubulin in rat colon formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab7291, 0.5ug/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  FABP4 (green) was detected using FABP4 primary antibody (Anti-FABP4 antibody [EPR3579] ab92501; diluted 1/1000). Alpha tubulin (red) was detected using the mouse monoclonal (ab7291) antibody. Cells were imaged by confocal microscopy, using z-stack for adipocyte-like cells.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Immunofluorescence staining of GABA Transporter 1 / GAT 1 using Anti-GABA Transporter 1 / GAT 1 antibody [EPR24202-20] ab259971 in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. The cells were fixed with 100% methanol (5 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 Anti-GABA Transporter 1 / GAT 1 antibody [EPR24202-20] ab259971 at 5 μg/ml and 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.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Immunofluorescent imaging of human cells (U2OS) with ab7291 reveals a delicate network of alpha-tubulin (green) located exclusively in the cytoplasm. The nucleus is stained blue.
  

  IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour. Primary antibody used at 1/200 in 5% milk / 0.2% TWEEN for one hour, secondary antibody Alexa 488 for 30 minutes. All blocking and incubation steps carried out at 37 degrees.

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Western blot: Anti-MGMT antibody [EPR4397] (Anti-MGMT antibody [EPR4397] ab108630) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-MGMT antibody [EPR4397] ab108630 was shown to bind specifically to MGMT. A band was observed at 22 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in MGMT CRISPR-Cas9 edited cell line Human MGMT knockout HCT116 cell line ab286541. The band observed in the CRISPR-Cas9 edited lysate lane below 22 kDa is likely to represent a truncated form of MGMT. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and MGMT CRISPR-Cas9 edited HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-MGMT antibody [EPR4397] (Anti-MGMT antibody [EPR4397] ab108630) at 1/1000 dilution

  Lane 1: Wild-type HCT 116 cell lysate at 20 µg

  Lane 2: Western blot - Human MGMT knockout HCT116 cell line (Human MGMT knockout HCT116 cell line ab286541)

  Lane 2: MGMT CRISPR-Cas9 edited HCT 116 cell lysate at 20 µg

  Secondary

  Lanes 1 - 2: Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

  Lanes 1 - 2: Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Observed band size: 22 kDa

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Western blot: Anti-FANCD2 antibody [EPR2302] (Anti-FANCD2 antibody [EPR2302] ab108928) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-FANCD2 antibody [EPR2302] ab108928 was shown to bind specifically to FANCD2. A band was observed at 160 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in FANCD2 knockout cell line. To generate this image, wild-type and FANCD2 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-FANCD2 antibody [EPR2302] (Anti-FANCD2 antibody [EPR2302] ab108928) at 1/1000 dilution

  Lane 1: Wild-type HCT 116 cell lysate at 20 µg

  Lane 2: Western blot - Human FANCD2 knockout HCT116 cell line (Human FANCD2 knockout HCT116 cell line ab286588)

  Lane 2: FANCD2 knockout HCT 116 cell lysate at 20 µg

  Lane 3: Wild-type HeLa ab255448 cell lysate at 20 µg

  Lane 4: FANCD2 knockout HeLa Human FANCD2 knockout HeLa cell line ab261743 cell lysate at 20 µg

  Secondary

  Lanes 1 - 4: Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

  Lanes 1 - 4: Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Observed band size: 160 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Alpha Tubulin Immunocytochemistry/ Immunofluorescence staining of Mouse splenocyte using mouse Anti-alpha Tubulin antibody

  Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Mouse splenocyte cells labelling CD18 with Anti-CD18 antibody [EPR29884-566] ab324101 at 1/50 (10.18 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

  Confocal image showing cytoplasmic staining in a subset of mouse splenocytes (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
  
  Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

  -ve control 1: Anti-CD18 antibody [EPR29884-566] ab324101 at 1/50 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) antibody at 1/1000 dilution.
  
  -ve control 2: ab7291 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution.

• 

  Western blot - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

  Western blot: Anti-PSPH antibody (Anti-PSPH antibody ab211418) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-PSPH antibody ab211418 was shown to bind specifically to PSPH. A band was observed at 25 kDa in wild-type A549 cell lysates with no signal observed at this size in PSPH knockout cell line. To generate this image, wild-type and PSPH knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-PSPH antibody (Anti-PSPH antibody ab211418) at 1/1000 dilution

  Lane 1: Wild-type A549 cell lysate at 20 µg

  Lane 2: PSPH knockout A549 cell lysate at 20 µg

  Lane 3: Caco-2 cell lysate at 20 µg

  Lane 4: A549 Nuclear cell lysate at 20 µg

  Secondary

  Lanes 1 - 4: Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

  Lanes 1 - 4: Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Observed band size: 25 kDa