Anti-beta III Tubulin 抗体 [EPR1568Y] (ab68193)

Flow cytometry overlay histogram showing wild-type HeLa (green line) and TUBB3 knockout HeLa stained with ab68193 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab68193) (1x 10⁶ in 100μl at 0.04 μg/ml (1/49250)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type HeLa - black line, TUBB3 knockout HeLa - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

ab68193 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 ab68193 at 1μg/ml concentration and ab7291, a mouse anti-tubulin antibody, at 1μg/ml overnight at +4°C. This is followed by a further incubation at room temperature for 1h with a goat anti-rabbit IgG Alexa Fluor® 488 (ab150081) at 2μg/ml (shown in green) and a goat anti-mouse IgG Alexa Fluor® 647 (ab150119) at 2μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.

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