Active Semaphorin 3A (SEMA3A)

The Semaphorin 3A(SEMA3A) which belongs to the semaphorin family can function as either a chemorepulsive agent, inhibiting axonal outgrowth, or as a chemoattractive agent, stimulating the growth of apical dendrites. In both cases, the protein is vital for normal neuronal pattern development. Semaphorin 3A is secreted protein containing a Sema domain, an immunoglobulin C2-like domain and a basic domain near the carboxyl tail. It can be secreted by neurons and surrounding tissue to guide migrating cells and axons in the developing nervous system. Besides, Neuropilin 1 (NRP1) has been identified as an interactor of SEMA3A, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant rat SEMA3A and recombinant human NRP1. Briefly, SEMA3A were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to NRP1-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-SEMA3A pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution , wells were incubated 15-25 minutes at 37℃. Finally, add 50 ul stop solution to the wells and read at 450 nm immediately. The binding activity of recombinant rat SEMA3A and recombinant human NRP1 was shown in Figure 1, the EC50 for this effect is 1.22 ug/mL.