ImagesImmunocytochemistry/ 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 actinknockout HAP1 cell lysate (20 µg)Lanes 1 and 2: Merged signal (red and green). Green - beta actin, ab6276observed at 42 kDa. Red - loading control, ab181602 observed at37 kDa.Ab6276 was shown to specifically react withbeta actinin wild-type HAP1 cells. No band was observed whenbeta actinknockout samples were used. Wild-type andbeta actinknockout 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 - Anti-beta Actin antibody [AC-15] (ab6276)This image is courtesy of an Abreview submitted by Dr Mark ElliottWestern 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).See AbreviewWestern blot - Anti-beta Actin antibody [AC-15] (ab6276)All lanes : Anti-beta Actin antibody [AC-15] (ab6276) at 1 µg/mlLane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysateLane 2 : Jurkat (human T cell leukemia cell line from peripheral blood) cell lysateLane 3 : COS-7 (african green monkey kidney fibroblast-like cell line) cell lysateLane 4 : NIH/3T3 (mouse embryonic fibroblast cell line) cell lysateLane 5 : PC-12 (rat adrenal gland pheochromocytoma cell line) cell lysateLane 6 : Rat2 (rat fibroblast cell line) cell lysateLane 7 : CHO (chinese hamster ovary cell line) cell lysateLane 8 : MDBK (bovine kidney cell line) cell lysateLane 9 : MDCK (canine kidney cell line) cell lysateSecondaryAll lanes : Goat Anti-Mouse IgG-PeroxidaseDeveloped using the ECL technique.Predicted band size: 42 kDaWestern blot - Anti-beta Actin antibody [AC-15] (ab6276)All lanes : Anti-beta Actin antibody [AC-15] (ab6276) at 1/5000 dilutionLane 1 : HeLa nuclearLane 2 : HeLa whole cell lysateLane 3 : A431 cell lysateLane 4 : Jurkat cell lysateLane 5 : HEK293 cell lysateLysates/proteins at 20 µg per lane.SecondaryAll lanes : Alexa Fluor anti mouse at 1/5000 dilutionPerformed under reducing conditions.Predicted band size: 42 kDaObserved band size: 42 kDaWestern blot - Anti-beta Actin antibody [AC-15] (ab6276)This image is courtesy of David Grimm, Yale University, USA.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.