Flow Cytometry (Intracellular) - Anti-alpha smooth muscle Actin antibody [1A4] (ab7817)

Overlay histogram showing SV40LT-SMC cells stained with ab7817 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab7817, 1.137μg/ml) for 30 min at 22°C. The secondary antibody used was Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113) at 1/2000 dilution for 30 min at 22°C

Isotype control antibody (black line) was mouse IgG2a [18C8BC7AD10] (ab170191) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.

Western blot - Anti-alpha smooth muscle Actin antibody [1A4] (ab7817)

Gel type: MOPS

Blocking buffer: 3% milk

Loading control: alpha tubulin (ab52866), secondary Goat anti-Rabbit IgG H&L (IRDye® 680CW) preadsorbed (1:10000 dilution)

All lanes:

Western blot - Anti-alpha smooth muscle Actin antibody [1A4] (ab7817) at 1 µg/mL

Lane 1:

NIH 3T3 whole cell lysate at 20 µg

Lane 2:

SV40LT-SMC whole cell lysate at 20 µg

Lane 3:

A431 whole cell lysate at 20 µg

Lane 4:

A549 whole cell lysate at 20 µg

Lane 5:

Jurkat whole cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Mouse IgG H&L (IRDye® 800RD) at 1/10000 dilution

Predicted band size: 42 kDa

Observed band size: 42 kDa

Immunocytochemistry/ Immunofluorescence - Anti-alpha smooth muscle Actin antibody [1A4] (ab7817)

ab7817 staining alpha smooth muscle Actin in SV40LT-SMC cells (positive control, top panel) and A431 cells (negative control, bottom panel). The cells were fixed with 100% methanol (5 min) then 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 ab7817 at 1μg/ml concentration and ab6046 (Rabbit polyclonal to beta Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor® 594) (ab150080) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.

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