Human PAM protein, His tag

Peptidyl-glycine alpha-amidating monooxygenase (PAM) is an enzyme that is required for the biosynthesis of many signaling peptides. This enzyme mainly includes two domains with distinct catalytic activities, a peptidylglycine alpha-hydroxylating monooxygenase (PHM) domain and a peptidyl-alpha- hydroxyglycine alpha-amidating lyase (PAL) domain. These catalytic domains work sequentially to catalyze neuroendocrine peptides to active alpha-amidated products. Besides, Glucosidase Alpha, Acid (GaA) has been identified as an interactor of PAM, thus a binding ELISA assay was conducted to detect the interaction of recombinant human PAM and recombinant human GaA. Briefly, PAM were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to GaA-coated microtiter wells and incubated for 2h at 37ºC. Wells were washed with PBST and incubated for 1h with anti-PAM 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 PAM and GaA was in a dose dependent manner.