The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 150 kDa (predicted molecular weight: 150 kDa).
Use a concentration of 5 µg/ml.
Use a concentration of 5 µg/ml.
Inositol 5-phosphatase which has a role in clathrin-mediated endocytosis.
Concentrated 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.
Belongs 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.
Binds 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.
Western blot - Synaptojanin antibody - Synaptic Marker (ab19904)
All lanes : Anti-Synaptojanin antibody - Synaptic Marker (ab19904) at 1 µg/ml
Lane 1 : Rat Brain lysate Lane 2 : Rat Brain lysate with Rat Synaptojanin peptide (ab20433) at 1 µg/ml
Lysates/proteins at 20 µg per lane.
Secondary Alexa Fluor Rabbit polyclonal to Goat IgG at 1/5000 dilution
Performed under reducing conditions.
Predicted band size : 150 kDa Observed band size : 150 kDa ab19904 detected the expected 150kDa band in non-boiled (rat brain lysate) sample only and the detection of this band was blocked by addition of immunising peptide ab20433
ICC/IF image of ab19904 stained rat PC12 cells. The cells were methanol fixed (5 min), permabilised in PBS-T (20 min) and incubated with the antibody (ab19904, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. 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). DAPI was used to stain the cell nuclei (blue).
Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Synaptojanin antibody - Synaptic Marker (ab19904)This image is courtesy of an abreview submitted by Sophie Pezet, CNRS, Paris, France
Immunohistochemistical detection of Synaptojanin using ab19904 on mouse brain PFA perfusion-fixed sections. Primary antibody ab19904 diluted at 1/300 and incubated for 18 hours @ 20°C in PBS + 0.3 % Triton X100. Secondary antibody: goat anti-rabbit Alexa Fluor 488® (1/1000). Image: immunostaining obtained in the mouse hippocampus on perfusion-fixed free floating sections using ab19904 at 1/300 using direct fluorescence. The staining is so strong that it is hard to determine the cellular localisation, but it seems to be cytoplasmic.
Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Synaptojanin antibody - Synaptic Marker (ab19904)Image from Wagnon JL et al., PLoS Genet 8(11): e1003067.. Fig 6.; doi: 10.1371/journal.pgen.1003067. Epub 2012 Nov 29.
Immunohistochemical analysis of PFA-perfusion fixed mouse brain tissue, staining Synaptojanin with ab19904.
Tissue was post-fixed in 4% paraformaldehyde overnight at 4°C. Sections were incubated in a blocking buffer of 0.3% Triton-X-100, 1% BSA and 10% NGS in PBS for 2 hours at room temperature, before incubating with primary antibody (1/100) for 2 days at 4°C. An AlexaFluor®-conjugated anti-rabbit IgG (1/1000) was used as the secondary antibody.
Synaptojanin was immunoprecipitated using 0.5mg Rat brain tissue, 5µg of Rabbit polyclonal to Synaptojanin and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Rat brain tissue lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab19904. Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Band: 150kDa; Synaptojanin
Wagnon JL et al. CELF4 regulates translation and local abundance of a vast set of mRNAs, including genes associated with regulation of synaptic function. PLoS Genet8:e1003067 (2012).
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