Sirtuin 4, also known as SIRT4, is a member of the sirtuin family. SIRT4 is a mitochondrial ADP-ribosyltransferase that inhibits mitochondrial glutamate dehydrogenase 1 activity, thereby downregulating insulin secretion in response to amino acids. It has been shown that SIRT4 regulates fatty acid oxidation and mitochondrial gene expression in liver and muscle cells. Besides, Heat Shock 60kD Protein 1, Chaperonin (HSPD1) has been identified as an interactor of SIRT4, thus a binding ELISA assay was conducted to detect the interaction of recombinant human SIRT4 and recombinant human HSPD1. Briefly, SIRT4 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μL were then transferred to HSPD1-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-SIRT4 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 SIRT4 and HSPD1 was shown in Figure 1, and this effect was in a dose dependent manner.
Figure. The binding activity of SIRT4 with HSPD1