Human PKM protein, His and GST tag

Pyruvate Kinase, Muscle (PKM2) is one of four isozymes of pyruvate kinase. In vertebrates there are four isozymes of pyruvate kinase: L (liver), R (erythrocytes), M1 (muscles, hearts and brain) and M2 (only form detectable in early fetal tissue and present in most adult tissues). Pyruvate kinase is the enzyme that catalyzes the final step of glycolysis. It catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP), yielding one molecule of pyruvate and one molecule of ATP. Besides, Peptidyl Prolyl Cis/Trans Isomerase NIMA Interacting Protein 1 (PIN1) has been identified as an interactor of PKM2, thus a binding ELISA assay was conducted to detect the interaction of recombinant human PKM2 and recombinant human PIN1. Briefly, PKM2 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to PIN1-coated microtiter wells and incubated for 2h at 37ºC. Wells were washed with PBST and incubated for 1h with anti-PKM2 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 PKM2 and PIN1 was in a dose dependent manner.