APRIL Antibody [Aprily-1]

Application Note:

APRIL antibody can be used to recognize human APRIL by Flow Cytometry (also works for intracellular FC staining) and Western Blot. Anti-APRIL (MAb Aprily-1) does not cross react with mouse APRIL. Method: HEK 293 cells were mock transfected or transfected with an expression plasmid enabling surface expression of hAPRIL. Cells (5x105) were incubated on ice for 30 min in 50 μl FACS buffer (PBS, 5% Fetal calf serum, 0.02% azide) containing 10 μg/mL of Aprily-1 antibody. After washing in FACS buffer, FITC-conjugated antibody to mouse IgG was added. Cells were incubated on ice for 30 min, washed and analyzed by flow cytometry. Method: Cell supernatants were first precleared with protein-A sepharose and then subjected to immunoprecipitation with rhBCMA:Fc and protein-A sepharose. Beads were washed twice with PBS. Immunoprecipitated materials were eluted with 2.5mM glycine pH 2.5 for 10 min, neutralized with Tris buffer and run on an SDS-PAGE gel and analyzed with 2 μg/mL Aprily-1 by Western blot. Note: In some migrations endogenous soluble APRIL resolved as a doublet around 20kDa (dendritic cells: DC). rhsMegaAPRIL served as a positive control. Method: 3 HEK 293T cells (5 x 105) were mock transfected (thin line) or transfected with an expression plasmid enabling surface expression of mDR6 (thick line). Cells were incubated on ice for 30 min in 50 μl FACS buffer (PBS, 5% Fetal calf serum, 0.02% azide) containing 1 μg/mL of Luke-1 antibody. After washing in FACS buffer, PE-conjugated antibody to rat IgG was added. Cells were incubated on ice for 30 min, washed and then analyzed by flow cytometry.