All lanes : Anti-Synaptojanin antibody - Synaptic Marker (ab19904) at 1 µg/mlLane 1 : Rat Brain lysateLane 2 : Rat Brain lysate with Rat Synaptojanin peptide (ab20433) at 1 µg/mlLysates/proteins at 20 µg per lane.SecondaryAlexa Fluor Rabbit polyclonal to Goat IgG at 1/5000 dilutionPerformed under reducing conditions.
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).
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.See Abreview
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