Bovine Insulin protein, His and GST tag

Insulin (INS) is a polypeptide hormone originating in the beta cells of the pancreas and serving as a principal regulator for the storage and production of carbohydrates. Insulin decreases blood glucose concentration. It increases cell permeability to monosaccharides, amino acids and fatty acids. It accelerates glycolysis, the pentose phosphate cycle, and glycogen synthesis in liver. Besides, Insulin Receptor (ISR) has been identified as an interactor of INS, thus a binding ELISA assay was conducted to detect the interaction of recombinant bovine INS and recombinant human ISR. Briefly, INS were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to ISR-coated microtiter wells and incubated for 2h at 37ºC. Wells were washed with PBST and incubated for 1h with anti-INS 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 of INS and ISR was in a dose dependent manner.