ab213243 staining beta-Actin in NIH3T3 cells. The cells were fixed with 100% methanol (5 min), 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 overnight at +4°C with ab213262 at 0.5ugml then detected with an Alexa Fluor^® 488 goat anti-rabbit secondary antibody (ab150081) at a 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue), and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at a 1/250 dilution (shown in red).

 

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

All lanes :

Lane 1 : HAP1 whole cell lysate at 20 µg
Lane 2 : HAP1 ACTNB knockout whole cell lysate at 20 µg
Lane 3 : MEF1 whole cell lysate at 20 µg
Lane 4 : NIH3T3 whole cell lysate at 20 µg
Lane 5 : Recombinant Human gamma Actin protein (ab157841) at 0.1 µg

Performed under reducing conditions.

Predicted band size: 42 kDa

ab213262 was shown to react with ACTNB (beta actin) when tested against HAP1 wild-type and HAP1 ACTNB (beta actin) knockout cells. ACTNB wild-type and knockout samples were subjected to SDS-PAGE and incubated with ab213262. Incomplete reduction of signal was seen in the ACTNB (beta actin) knockout samples at beta actin's expected molecular weight. SDS-PAGE of gamma actin recombinant protein and incubation with ab213262 showed cross-reactivity to gamma actin.

ab213262 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1ug/ml and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ab213243 staining beta-Actin in HeLa cells. The cells were fixed with 100% methanol (5 min), 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 overnight at +4°C with ab213262 at 0.01ugml then detected with an Alexa Fluor^® 488 goat anti-rabbit secondary antibody (ab150081) at a 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue), and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at a 1/250 dilution (shown in red).

 

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