The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
1/100 - 1/500. Detects a band of approximately 48 kDa (predicted molecular weight: 48 kDa).
Members of the STE-20 like kinase family are known to stimulate MAPK pathways by directly activating MAPKKK. LYK5 is a novel pseudokinase member of this family consisting of a STE-20 like kinase domain but lacks several residues that are required for its catalytic activity. It specifically binds LKB1 and plays a key role in regulating the tumor suppressor activities of LKB1. It functions as an upstream activator of LKB1 and also directs the sub-cellular localization of LKB1 by anchoring it in the cytoplasm. LYK5-LKB1 interaction results in phosphorylation of LYK5 and enhanced autophosphorylation of LKB1.
ab71476 stained HepG2 cells. The cells were 4% formaldehyde 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 ab71476 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) 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.
Western blot - LYK5 antibody - N-terminal (ab71476)
Anti-LYK5 antibody (ab71476) at 1/100 dilution + mouse brain tissue lysate at 12.5 µg
Predicted band size: 48 kDa Observed band size: 48 kDa Additional bands at: 60 kDa. We are unsure as to the identity of these extra bands.
Asada N & Sanada K LKB1-mediated spatial control of GSK3beta and adenomatous polyposis coli contributes to centrosomal forward movement and neuronal migration in the developing neocortex. J Neurosci30:8852-65 (2010).
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