ImagesWestern blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)This image is courtesy of an anonymous AbreviewAll lanes : Anti-GAPDH antibody [6C5] - Loading Control (ab8245)Lane 1 : Mouse hippocampus whole cell lysateLane 2 : Rat hippocampus whole cell lysateLysates/proteins at 20 µg per lane.SecondaryAll lanes : HRP-conjugated Rabbit anti-mouse at 1/5000 dilutionDeveloped using the ECL technique.Performed under reducing conditions.Predicted band size: 40.2 kDaObserved band size: 36 kDa why is the actual band size different from the predicted?Exposure time: 10 secondsSee AbreviewImmunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)ab8245 staining GAPDH in SV40LT-SMC (RatSV40-transfected aorta smooth cell line) cells.The cells were fixed with 4% formaldehyde (10 minutes),permeabilized with 0.1% Triton X-100 for 5 minutes andthen blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for1 hour. The cells were then incubated with ab8245 at 5μg/ml and ab202272 at 1/250 overnight at +4°C, followed by a further incubation at room temperature for 1h withGoat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117)(shown in green). Nuclear DNA was labeled in blue with DAPI.Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).Western blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 10 µg/ml + Raji (Human Burkitt"s lymphoma cell line) whole cell lysate at 20 µgPredicted band size: 40.2 kDaWestern blot - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)All lanes : Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 2.5 µg/mlLane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) NuclearLane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysateLane 3 : A431 (Human epidermoid carcinoma cell line) cell lysateLane 4 : Jurkat (Human T cell leukemia cell line from peripheral blood) cell lysateLane 5 : HEK-293 (Human epithelial cell line from embryonic kidney) cell lysateLysates/proteins at 20 µg per lane.SecondaryAll lanes : Alexa Fluor anti-mouse at 1/5000 dilutionPerformed under reducing conditions.Predicted band size: 40.2 kDaObserved band size: 37 kDa why is the actual band size different from the predicted?Fluorescence detection of secondary antibody.Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)ab8245 staining GAPDH in NIH/3T3 (Mouse embryo fibroblast cell line) cells.The cells were fixed with 4% formaldehyde (10 minutes) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1 hour. The cells were then incubated with ab8245 at 1 μg/ml and ab202272 at 1/250 overnight at +4°C, followed by a further incubation at room temperature for 1 hour withGoat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117)(shown in green). Nuclear DNA was labeled in blue with DAPI.Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [6C5] - Loading Control (ab8245)ab8245 stainingGAPDH in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.The cells were fixed with 100% methanol (5 minutes) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1 hour. The cells were then incubated with ab8245 at 5 μg/ml and ab6046 at 1 µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1 hour withGoat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117)at 2 μg/ml (shown in green) andGoat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (ab150088)at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labeled in blue with DAPI.Negative controls: 1– Rabbit primary antibodyand anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.