Western blot analysis of Semaphorin 4A expression in HEK293T (A), A549 (B), SGC7901 (C) whole cell lysates. (Predicted band size: 83 kD; Observed band size: 95 kD)

Immunohistochemical analysis of Semaphorin 4A staining in human brain formalin fixed paraffin embedded tissue section. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0). The section was then incubated with the antibody at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

Immunofluorescent analysis of Semaphorin 4A staining in HeLa cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with the primary antibody in 3% BSA-PBS and incubated overnight at 4 °C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight 594-conjugated secondary antibody (red) in PBS at room temperature in the dark. DAPI was used to stain the cell nuclei (blue).

Immunoprecipitation of Semaphorin 4A from 0.5mg Jurkat whole cell extract lysate, using 5ug of Anti-Semaphorin 4A Antibody and 50ul of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Jurkat whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation. Proteins were eluted by addition of 40ul SDS loading buffer and incubated for 10min at 70°C; 10ul of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with Anti-Semaphorin 4A Antibody.