The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 10 µg/ml.
Use a concentration of 1 µg/ml. Detects a band of approximately 21 kDa (predicted molecular weight: 21 kDa).
Plasma membrane-associated small GTPase which cycles between an active GTP-bound and an inactive GDP-bound state. In active state binds to a variety of effector proteins to regulate cellular responses. Involved in epithelial cell polarization processes. Causes the formation of thin, actin-rich surface projections called filopodia.
Belongs to the small GTPase superfamily. Rho family. CDC42 subfamily.
AMPylation at Tyr-32 and Thr-35 are mediated by bacterial enzymes in case of infection by H.somnus and V.parahaemolyticus, respectively. AMPylation occurs in the effector region and leads to inactivation of the GTPase activity by preventing the interaction with downstream effectors, thereby inhibiting actin assembly in infected cells. It is unclear whether some human enzyme mediates AMPylation; FICD has such ability in vitro but additional experiments remain to be done to confirm results in vivo.
Lane 1: Wild-type HAP1 cell lysate (20 µg) Lane 2: CDC42 knockout HAP1 cell lysate (20 µg) Lane 3: MCF7 cell lysate (20 µg) Lane 4: HepG2 cell lysate (20 µg) Lanes 1 - 4: Merged signal (red and green). Green - ab66573 observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab66573 was shown to specifically react with CDC42 in wild-type HAP1 cells. No band was observed when CDC42 knockout samples were examined. Wild-type and CDC42 knockout samples were subjected to SDS-PAGE. ab66573 and ab8245 (loading control to GAPDH) were diluted at 1µg/ml and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.
Western blot - CDC42 antibody (ab66573)
All lanes : Anti-CDC42 antibody (ab66573) at 1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate Lane 4 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate Lane 5 : U2OS (Human osteosarcoma cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Performed under reducing conditions.
Predicted band size: 21 kDa Observed band size: 21 kDa Additional bands at: 42 kDa, 48 kDa, 52 kDa. We are unsure as to the identity of these extra bands.
ICC/IF image of ab66573 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab66573, 10µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899 Dylight 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.