Immunocytochemistry/ Immunofluorescence - Anti-beta Actin antibody [AC-15] (ab6276)

ab6276 staining beta Actin in SV40LT-SMC cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab6276 at a working concentration of 5μg/ml and ab190573, Rabbit monoclonal [EP1332Y] to alpha Tubulin (Alexa Fluor® 647, shown in red) at 1/250 overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-mouse AlexaFluor® 488 (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

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

Western blot - Anti-beta Actin antibody [AC-15] (ab6276)

Lane 1: Wild-type HAP1 cell lysate (20 μg)

Lane 2: Beta actin knockout HAP1 cell lysate (20 μg)

Lanes 1 and 2: Merged signal (red and green). Green - beta actin, ab6276 observed at 42 kDa. Red - loading control, ab181602 observed at 37 kDa.

ab6276 was shown to specifically react with beta actin in wild-type HAP1 cells. No band was observed when beta actin knockout samples were used. Wild-type and beta actin knockout samples were subjected to SDS-PAGE. ab6276 (beta actin) and ab181602 (loading control to GAPDH) were diluted 1/5000 and 1/10 000 and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

Lanes 1 - 2:

Western blot - Anti-beta Actin antibody [AC-15] (ab6276) at 1/5000 dilution

Lanes 1 - 2:

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) at 1/10000 dilution

Lane 1:

Wild-type HAP1 cell lysate (20 µg)

Lane 2:

Beta actin knockout HAP1 cell lysate (20 µg)

Secondary

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) at 1/10000 dilution

Lanes 1 - 2:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) at 1/10000 dilution

Predicted band size: 36 kDa, 41 kDa

Western blot - Anti-beta Actin antibody [AC-15] (ab6276)

All lanes:

Western blot - Anti-beta Actin antibody [AC-15] (ab6276) at 1 µg/mL

Lane 1:

HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate

Lane 2:

Jurkat (human T cell leukemia cell line from peripheral blood) cell lysate

Lane 3:

COS-7 (african green monkey kidney fibroblast-like cell line) cell lysate

Lane 4:

NIH/3T3 (mouse embryonic fibroblast cell line) cell lysate

Lane 5:

PC-12 (rat adrenal gland pheochromocytoma cell line) cell lysate

Lane 6:

Rat2 (rat fibroblast cell line) cell lysate

Lane 7:

CHO (chinese hamster ovary cell line) cell lysate

Lane 8:

MDBK (bovine kidney cell line) cell lysate

Lane 9:

MDCK (canine kidney cell line) cell lysate

Secondary

All lanes:

Goat Anti-Mouse IgG-Peroxidase

Developed using the ECL technique.

Predicted band size: 41 kDa

Western blot - Anti-beta Actin antibody [AC-15] (ab6276)

All lanes:

Western blot - Anti-beta Actin antibody [AC-15] (ab6276) at 1/5000 dilution

Lane 1:

HeLa nuclear at 20 µg

Lane 2:

HeLa whole cell lysate at 20 µg

Lane 3:

A431 cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Lane 5:

HEK293 cell lysate at 20 µg

Secondary

All lanes:

Alexa Fluor anti mouse at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 41 kDa

Observed band size: 42 kDa