Mouse NUCB2 protein, His and GST tag

Nucb2 (Nucleobindin-2), a calcium-binding protein, is further cleaved into NES1 (Nesfatin-1). NES1 is an anorexigenic peptide and seems to participate in hypothalamic pathways regulating food intake and energy homeostasis, acting in a leptin-independent manner. GADD45A (Growth arrest and DNA damage-inducible protein GADD45 alpha) has been identified as an interactor of Nucb2. Besides, rat GADD45A shares similarities with mouse NES1 in amino acid sequence with the identity of 98% . Thus a binding ELISA assay was conducted to detect the interaction of recombinant mouse NES1 and recombinant rat GADD45A. Briefly, NES1 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl NES1 were then transferred to GADD45A-coated microtiter wells and incubated for 2h at 37ºC. Wells were washed with PBST and incubated for 1h with anti-NES1 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 37ºC. Finally, add 50 μl stop solution to the wells and read at 450nm immediately. The binding activity of NES1 and GADD45A was in a dose dependent manner.