Cold-inducible RNA-binding protein (CIRBP) plays a critical role in controlling the cellular response upon confronting a variety of cellular stresses, including short wavelength ultraviolet light, hypoxia, and hypothermia. Besides, Heterogeneous Nuclear Ribonucleoprotein A1 (HNRPA1) has been identified as an interactor of CIRBP, thus a binding ELISA assay was conducted to detect the interaction of recombinant mouse CIRBP and recombinant mouse HNRPA1. Briefly, CIRBP were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μL were then transferred to HNRPA1-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-CIRBP 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 CIRBP and HNRPA1 was shown in Figure 1, and this effect was in a dose dependent manner.
Figure. The binding activity of CIRBP with HNRPA1.