The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use a concentration of 1 µg/ml. Detects a band of approximately 119 kDa (predicted molecular weight: 119 kDa).
FunctionComponent of clathrin-coated pits and vesicles, that may link the endocytic machinery to the actin cytoskeleton. Binds 3-phosphoinositides (via ENTH domain). May act through the ENTH domain to promote cell survival by stabilizing receptor tyrosine kinases following ligand-induced endocytosis.
Tissue specificityBrain, heart, kidney, pancreas, and liver, but not in lung or placenta.
Sequence similaritiesBelongs to the SLA2 family. Contains 1 ENTH (epsin N-terminal homology) domain. Contains 1 I/LWEQ domain.
DomainBinds F-actin via the talin-like I/LWEQ domain.
Cellular localizationCytoplasm > perinuclear region. Endomembrane system. Cytoplasmic vesicle > clathrin-coated vesicle membrane. Membrane-associated protein, mainly localized at the endocytic compartments and in the perinuclear region.
ICC/IF image of ab77297 stained PC12 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 (ab77297, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 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. This antibody also gave a positive result in 100% methanol fixed (5 min) PC12 cells at 5µg/ml.
Xenopus laevis cytoplasmic egg extract visualized live with primary and secondary antibody addition [red is anti-Clathrin X22 (ab2731) with goat anti-mouse Alexa Fluor 568 secondary, green is anti-HIP1R (ab77297) with goat anti-rabbit Alexa Fluor 488 secondary]. HIP1R staining is specific for perinuclear membrane compartments.