Anti-beta Actin antibody [AC-15] - Loading Control

• WB

Lab

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

Lanes 1 - 2:

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (<a href='/en-us/products/primary-antibodies/gapdh-antibody-epr16891-loading-control-ab181602'>ab181602</a>) 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 (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-800cw-preadsorbed-ab216772'>ab216772</a>) at 1/10000 dilution

Lanes 1 - 2:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 36 kDa,41 kDa

false

• WB

AbReview10565****

Western blot - Anti-beta Actin antibody [AC-15] - Loading Control (AB6276)

Western Blot of ab6276 (used as loading control) with whole tissue lysate of human 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).

All lanes:

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

Predicted band size: 41 kDa

Observed band size: 42 kDa

false

This image is courtesy of an Abreview submitted by Dr Mark Elliott

• WB

Supplier Data

Western blot - Anti-beta Actin antibody [AC-15] - Loading Control (AB6276)

All lanes:

Western blot - Anti-beta Actin antibody [AC-15] - Loading Control (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

Predicted band size: 41 kDa

true

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-beta Actin antibody [AC-15] - Loading Control (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).

• WB

Unknown

Western blot - Anti-beta Actin antibody [AC-15] - Loading Control (AB6276)

All lanes:

Western blot - Anti-beta Actin antibody [AC-15] - Loading Control (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

Predicted band size: 41 kDa

Observed band size: 42 kDa

false

• WB

AbReview225****

Western blot - Anti-beta Actin antibody [AC-15] - Loading Control (AB6276)

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.

All lanes:

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

Predicted band size: 41 kDa

false

This image is courtesy of David Grimm, Yale University, USA.