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. Predicted molecular weight: 144 kDa. Good results were obtained when blocked with 5% non-fat dry milk in 0.05% PBS-T.
FunctionInositol 5-phosphatase which has a role in clathrin-mediated endocytosis.
Tissue specificityConcentrated at clathrin-coated endocytic intermediates in nerve terminals. Isoform 1 is more enriched than isoform 2 in developing brain as well as non-neuronal cells. Isoform 2 is very abundant in nerve terminals.
Sequence similaritiesBelongs to the synaptojanin family. In the central section; belongs to the inositol-1,4,5-trisphosphate 5-phosphatase family. Contains 1 RRM (RNA recognition motif) domain. Contains 1 SAC domain.
DomainBinds to EPS15 (a clathrin coat-associated protein) via a C-terminal domain containing three Asn-Pro-Phe (NPF) repeats. The C-terminal proline-rich region mediates binding to a variety of SH3 domain-containing proteins including AMPH, SH3GL1, SH3GL2, SH3GL3 and GRB2.
ab84309 stained SKNSH cells. The cells were 4% formaldhyde 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 (ab84309 at 5µg/ml) 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 - Synaptojanin antibody (ab84309)
Anti-Synaptojanin antibody (ab84309) at 1 µg/ml (in 5% skim milk / PBS buffer) + HepG2 cell lysate at 10 µg
Secondary HRP conjugated anti-Rabbit IgG at 1/50000 dilution