Growth arrest and DNA-damage-inducible protein GADD45 alpha (GADD45a) is a member of the GADD45 family. It may affect PCNA interaction with some CDK (cell division protein kinase) complexes; stimulates DNA excision repair in vitro and inhibits entry of cells into S phase. In T-cells, functions as a regulator of p38 MAPKs by inhibiting p88 phosphorylation and activity. Besides, Proliferating Cell Nuclear Antigen (PCNA) has been identified as an interactor of GADD45a, thus a binding ELISA assay was conducted to detect the interaction of recombinant rat GADD45a and recombinant rat PCNA. Briefly, GADD45a were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μL were then transferred to PCNA-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-GADD45a 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 50µL stop solution to the wells and read at 450nm immediately. The binding activity of GADD45a and PCNA was shown in Figure 1, and this effect was in a dose dependent manner.
Figure. The binding activity of GADD45a with PCNA.