APRIL Antibody [Aprily-8]

Application Note:

APRIL antibody can be used to recognize human APRIL by Flow Cytometry, immunocytochemistry and Western Blot. Method: 3 days after transfection of cells with the indicated constructs, cells were fixed with 4% formaldehyde 5 min at RT. After a wash in PBS, samples were dehydrated by washes in 60%, 80%, 90%, 100% EtOH and xylol. Cells were then dried and embedded in paraffin. Sections were cut, mounted on slides and dried overnight at 50°C. Slides were then successively washed 2x 10min in xylol, 2x 10min in 100% EtOH, and then treated 10 min in 100% MeOH/ 0.6% H202 to inhibit endogenous peroxidase. Samples were rehydrated by washes in 90%, 80%, 60% EtOH and PBS. After micro-wave treatment, slides were washed 3x in PBS, blocked with IgG, and incubated for 1 hour with 5 μg/mL Aprily-8 or control mouse IgG (isotype control) in 1%BSA / 1x PBS for 1 hour. After PBS washes, samples were incubated with the secondary Ab for 1 hour, washed in PBS and revealed with StreptABComplex/HRP (Vector) and AEC. Method: HEK 293 cells were mock transfected or transfected with an expression plasmid coding for human APRIL. 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-8 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: 10 μg of protein was applied to the gel, and revealed with Aprily-8 (1 μg/mL) and HRP-coupled anti-mouse secondary antibody (1:4,000).Note: The hAPRIL construct used in this experiment is an uncleavable fusion protein between hBAFF (aa 1-132) and hAPRIL (aa 93-233). This product is for research use only.