Alkaline Phosphatase (ALP) belongs to the group of metalloenzymes and can be found in prokaryotic and higher eukaryotic cells. These glycoproteins are active as dimers and hydrolyze phosphoric acid ester to alcohol and phosphate. Next to metal cofactors (Zn2+ and Mg2+) an alkaline milieu (pH 9.8) is required, since an excess of free protons would inhibit the enzymatic reaction.
Various chromogenic substrates can be hydrolyzed by ALP and turned into colorful precipitates (see table). ALP is suited as reporter molecule for different immunoassays. Alkaline phosphatase conjugates for secondary antibodies from Jackson Immunoresearch are isolated from calf intestines and prepared by a modified protocol (Alvarez et al., 1978). These conjugates contain heterogenic complexes with a rather high molecular weight (86 kDa) that can limit tissue-penetration, making ALP-conjugated secondary antibodies not the first choice for Immunohistochemistry (IHC).
The highly sensitive phosphates is ideal for solid-phase immunoassays (Western Blot, ELISA). Unlike other enzyme-conjugates substrate-converting reaction is linear, allowing higher sensitivity with extended incubation time. In addition, less problems occur with unspecific background staining since ALP-conjugates are suited for tissues with a high level of endogenic peroxidase.
Commonly used Substrates:
|5-bromo-4-chloro-3-indolyl phosphate (BCIP) / nitro blue tetrazolium (NBT)||Blue/ purple|
|Fast Red||Red/ orange|
|Neufuchsin||magenta (fuchsin red)|
You can find chromogenes and substrates here.
- Jackson ImmunoResearch: Catalogue 2019
- Sharma, U., Pal, D. & Prasad, R. Alkaline Phosphatase: An Overview. Indian J Clin Biochem 29, 269–278 (2014).
- Jackson ImmunoResearch: Chromogenic Detection for Western Blot, IHC, and ELISA (Jul 13 16)