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).

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.

Western Blot of ab6276 (used as loading control) with whole tissue lysate of grey matter from BA20 (temporal cortex).  Ab6276 was diluted 1/50000 and incubated with the sample for 16 hours at 4°C.  5 µg of lysate was loaded onto the gel, which was blocked with 5% milk for 1 hour at 20°C.  An Alexa Fluor® 680 conjugated goat anti-mouse antibody, diluted 1/5000, was used as the secondary.

Bands below actin in image are synaptophysin (SYN).

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All lanes : 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: 42 kDa

All lanes : Anti-beta Actin antibody [AC-15] (ab6276) at 1/5000 dilution

Lane 1 : HeLa nuclear
Lane 2 : HeLa whole cell lysate
Lane 3 : A431 cell lysate
Lane 4 : Jurkat cell lysate
Lane 5 : HEK293 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Alexa Fluor anti mouse at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 42 kDa
Observed band size: 42 kDa

MDCK cells induced with increasing amounts of doxycycline to control expression of the gene of interest. All cells were normalized for loading with an albumin protein standard assay. Anti-beta actin (ab6276) was used at a concentration of 1:5000 in a milk blocking solution. B-actin blotting confirms the albumin assay in showing that an equal amount of lysate was loaded in each lane.