Alkaline Phosphatase ImmunoAssays
Alkaline Phosphatase (AP), like Horse Radish Peroxidase (HRP), is widely used in classical protein assays, mainly immunoassays like ELISA and Western blotting. The enzyme is typically conjugated to a secondary antibody, and in combination to an appropriate substrate, it allows amplifying an antigen-antibody binding signal.
Three Convenient Assay Modes:
- Fluorimetry using a highly purified FDP substrate which emits in the red channel (ex/em = 545/590 nm). In the ELISA assay formats, the kits provide an Alkaline phosphatase-conjugated secondary antibody (goat anti-mouse IgG or goat-anti-rabbit IgG)
- Luminometry using an optimal chemiluminescent substrate for superior signal-to-noise ratio
- Colorimetry, where the classical pNPP substrate allows signal monitoring at at 450 nm
Adapted to various samples: detection of AP in solution, cell extracts, live cells as well as on solid surfaces (such as PVDF membranes).
‘Mix and read’ assay protocol compatible with HTS liquid handling instruments for the bulk assay formats
Alkaline Phosphatase Reporter Gene Assays
Secreted alkaline phosphatase (SEAP) is widely used as a reporter gene to analyze gene expression, the activity of promoters and transcriptional factors in cell culture or animals.
Our SensoLyte® Secreted Alkaline Phosphatase Reporter Gene Assays provide sensitive assays of placental alkaline phosphatase for both secreted and membrane-bound forms of the enzyme. Our two kits allow a detection in colorimetry (using pNPP; detection at 405 nm) or chemiluminescence. Both kits are based on a “mix and read” assay protocol that is compatible with high throughput screening.
The change of acid phosphatase expression level and activity is involved in a variety of physiological and pathological events, such as prostate puberty, rheumatoid arthritis, bone-resorption related diseases, serum marker of tumor bone metastasis, and diabetes. Our SensoLyte® Assay Kit is based on the fluorescence of the MFP hydrolyzed product which is pH-insensitive and can be used to continuously monitor phosphatase activity in acidicic conditions.
More about Phophatases
Phophatases hydrolyze a variety of organic phosphate monoesters to alcohols and inorganic phosphate. This important class of enzymes is involved in cellular signal transduction, cell metabolism and other biological activities. Some phosphatases serve as enzyme markers. For example, PALP-1, the gene for human placental alkaline phosphate, has been used as a eukaryotic receptor gene that is superior to IacZ for lineage studies in murine retina. This gene has also been engineered to produce a secreted alkaline phosphatase (SEAP), allowing quantitation of gene expression without disrupting the cells. Some phosphatases (such as protein phosphatase) are identified as drug screening targets.