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Tissue/ cell preparation - acetylated alpha-tubulin from the axoneme of sea urchin sperm flagella.
This antibody binds to primary cilia, centrioles, mitotic spindles, midbodies and to subsets of cytoplasmic microtubules in 3T3 and HeLa cells.
Our Abpromise guarantee covers the use of ab24610 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||Use 1µl for 106 cells.|
|IP||Use at an assay dependent concentration.|
|WB||1/1000. Detects a band of approximately 55 kDa (predicted molecular weight: 55 kDa).|
|ICC||Use a concentration of 1 µg/ml.|
|IHC-P||1/100. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|ICC/IF||1/200 - 1/1000.|
ab24610 staining Acetylated alpha Tubulin in monkey kidney cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 3% PFA + 0.1% GA and blocked with 3% BSA + 0.5% Triton X-100 for 45 minutes at 25°C. Samples were incubated with primary antibody (1/100 in 3% BSA + 0.5% Triton X-100) for 1 hour at 21°C. An Alexa Fluor® 647-conjugated donkey anti-rabbit IgG polyclonal (2 µg/ml) was used as the secondary antibody.
ICC/IF image of ab24610 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab24610, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).
Overlay histogram showing HeLa cells stained with ab24610 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab24610, 1µg/1x106 cells) for 30 min at 22ºC. (This data was generated from a purified version of the antibody. Some lots are produced as ascites fluid. We suggest 1µl/1x106 cells for ascites preparations). The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
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