Immunohistochemistry (Formalin/PFa-fixed paraffin-embedded sections) analysis of human brain tissue labelling PAK5 with ab37753. A peroxidase-conjugated anti-rabbit IgG was used as the secondary antibody, followed by DAB staining.
ab37753 stained SKNSH cells. The cells were 4% formaldehyde fixed for 10 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab37753 at 1/50 dilution) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
Western blot - Anti-PAK5 antibody (ab37753)
All lanes : Anti-PAK5 antibody (ab37753) at 1/100 dilution
Lane 1 : COS7 cells (negative control) Lane 2 : transiently transfected COS7 cells with PAK1 Lane 3 : transiently transfected COS7 cells with PAK2 Lane 4 : transiently transfected COS7 cells with PAK4 Lane 5 : transiently transfected COS7 cells with PAK5 Lane 6 : transiently transfected COS7 cells with PAK6
Predicted band size: 81 kDa Observed band size: 81 kDa Additional bands at: 37 kDa, 50 kDa, 65 kDa. We are unsure as to the identity of these extra bands.