Anti-alpha Tubulin antibody [TU-01] - Loading Control

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-alpha Tubulin antibody [TU-01] - Loading Control (AB7750)

Overlay histogram showing HEK293 cells stained with ab7750 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton 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 (ab7750, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was anti-mouse DyLight® 488 (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK293 cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Triton used under the same conditions.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-alpha Tubulin antibody [TU-01] - Loading Control (AB7750)

Separation of HeLa cells stained using ab7750 (concentration in sample 3 μg/ml, GAM APC, red-filled) from HeLa cells unstained by primary antibody (GAM APC, black-dashed) in flow cytometry analysis (intracellular staining).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [TU-01] - Loading Control (AB7750)

ICC/IF image of ab7750 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab7750, 5μg/ml) overnight at +4°C. The secondary antibody (green) was anti-mouse DyLight® 488 (ab96879) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha Tubulin antibody [TU-01] - Loading Control (AB7750)

ab7750 staining human normal skin. Staining is localised to the cytoplasm.
Left panel : with primary antibody at 2 ug/ml. Right panel : isotype control.
Sections were stained using an automated system at room temperature. Sections were rehydrated and antigen retrieved with a retrieval buffer EDTA pH 9.0 . Slides were peroxidase blocked in 3% H₂O₂ in methanol for 10 minutes. They were then blocked for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with an amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [TU-01] - Loading Control (AB7750)

Immunocytochemistry analysis of HeLa (human cervix carcinoma) cells labelling alpha tubulin (red) with ab7750. DAPI was used to stain the nuclei blue.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [TU-01] - Loading Control (AB7750)

Immunocytochemistry/ Immunofluorescence analysis of NIH 3T3 (mouse embryonal fibroblast) cells labelling alpha tubulin (green) with ab7750. Vimentin was stained red as counterstain. DAPI was used to stain the nuclei blue.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody [TU-01] - Loading Control (AB7750)

Immunocytochemistry analysis of NIH 3T3 (mouse embryonal fibroblast) cells labelling alpha tubulin (green) with ab7750. DAPI was used to stain the nuclei blue.

• WB

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Western blot - Anti-alpha Tubulin antibody [TU-01] - Loading Control (AB7750)

Western blotting analysis labeling alpha-tubulin using ab7750 on lysates of various cell lines under reducing and non-reducing conditions. Nitrocellulose membrane was probed with 2 μg/ml of ab74696 followed by IRDye800-conjugated streptavidin. A specific band was detected for alpha-tubulin at approximately 54 kDa.

All lanes:

Western blot - Anti-alpha Tubulin antibody [TU-01] - Loading Control (ab7750)

Predicted band size: 50 kDa

Observed band size: 54 kDa

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