PAM ELISA Kit (Human)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level Peptidylglycine Alpha Amidating Monooxygenase (PAM) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level Peptidylglycine Alpha Amidating Monooxygenase (PAM) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

PAL; pancreatic peptidylglycine alpha-amidating monooxygenase; peptidyl alpha-amidating enzyme; peptidyl-alpha-hydroxyglycine alpha-amidating lyase; peptidylamidoglycolate lyase; peptidylglycine 2-hydroxylase; peptidyl-glycine alpha-amidating monooxygenase; peptidylglycine alpha-hydroxylating monooxygenase; PHM.

Bifunctional enzyme that catalyzes 2 sequential steps in C-terminal alpha-amidation of peptides. The monooxygenase part produces an unstable peptidyl (2-hydroxyglycine) intermediate that is dismutated to glyoxylate and the corresponding desglycine peptide amide by the lyase part. C-terminal amidation of peptides such as neuropeptides is essential for full biological activity.

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Peptidylglycine Alpha Amidating Monooxygenase (PAM) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Peptidylglycine Alpha Amidating Monooxygenase (PAM) . Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Peptidylglycine Alpha Amidating Monooxygenase (PAM), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm +/- 10nm. The concentration of Peptidylglycine Alpha Amidating Monooxygenase (PAM) in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Reconstitution & Storage Instructions Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Antibody Array (AA) Protocol

Matrix Recovery Range (%) Average (%) serum (n=5) 97-105 101 EDTA plasma (n=5) 86-96 93 heparin plasma (n=5) 87-95 90 Matrix Recovery Range (%) Average (%) Matrix Recovery Range (%) Average (%) serum (n=5) 97-105 101 serum (n=5) 97-105 101 EDTA plasma (n=5) 86-96 93 EDTA plasma (n=5) 86-96 93 heparin plasma (n=5) 87-95 90 heparin plasma (n=5) 87-95 90

Component Amount Anti-PAM Microplate 96 Wells (12 x 8 Well Strips) PAM Lyophilized Standard 2 x 2 ng 100X Biotinylated PAM Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Component Amount Component Amount Anti-PAM Microplate 96 Wells (12 x 8 Well Strips) Anti-PAM Microplate 96 Wells (12 x 8 Well Strips) PAM Lyophilized Standard 2 x 2 ng PAM Lyophilized Standard 2 x 2 ng 100X Biotinylated PAM Detector Antibody 120 uL 100X Biotinylated PAM Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Stop Solution 1 x 6 mL

This assay has high sensitivity and excellent specificity for detection of Peptidylglycine Alpha Amidating Monooxygenase (PAM) . No significant cross-reactivity or interference between Peptidylglycine Alpha Amidating Monooxygenase (PAM) and analogues was observed.