Alkaline Phosphatase (AP)
Alkaline Phosphatase is an important tool in molecular biological processes like cloning. It removes 3’- phosphate groups from a variety of substrates. Although in laboratory, it is used to catalyze the removal of terminal 5’-(P), residues from single stranded or double stranded DNA and RNA. The resulting 5’-OH termini can no longer take part in ligation reactions, thus prevents self religation of vectors, reducing the background of transformed bacterial colonies that carry empty plasmids. This enzyme works optimally at alkaline pH (range of 89 in the presence of low Zn+2 concentrations) and hence derived the name. Alkaline Phosphatase is isolated from various sources:-
a) Bacterial Alkaline phosphatase Secreted in monomeric form into the Periplasmic space of E.coli, where it form dimers and gets catalytically activated. It’s a remarkably stable enzyme and is resistant to inactivation by heat and detergent. Thus, bacterial alkaline phosphatase is the most difficult to destroy in the reaction mix.
b) Calf Intestinal Phosphatase Calf intestinal phosphatase is a dimeric glycoprotein isolated from bovine intestine. This has much more practical significance than bacterial alkaline phosphatase, since it can be readily inactivated from the reaction mixture using proteinase K or by heating at 650C for 30 minutes or 750C for 15 minutes in the presence of 10mM EGTA.
c) Shrimp alkaline phosphatase Extracted from cold water shrimp, can be inactivated readily by heating at 650C for 15 min.
Alkaline Phosphatase is an important tool in molecular biological processes like cloning. It removes 3’- phosphate groups from a variety of substrates. Although in laboratory, it is used to catalyze the removal of terminal 5’-(P), residues from single stranded or double stranded DNA and RNA. The resulting 5’-OH termini can no longer take part in ligation reactions, thus prevents self religation of vectors, reducing the background of transformed bacterial colonies that carry empty plasmids. This enzyme works optimally at alkaline pH (range of 89 in the presence of low Zn+2 concentrations) and hence derived the name. Alkaline Phosphatase is isolated from various sources:-
a) Bacterial Alkaline phosphatase Secreted in monomeric form into the Periplasmic space of E.coli, where it form dimers and gets catalytically activated. It’s a remarkably stable enzyme and is resistant to inactivation by heat and detergent. Thus, bacterial alkaline phosphatase is the most difficult to destroy in the reaction mix.
b) Calf Intestinal Phosphatase Calf intestinal phosphatase is a dimeric glycoprotein isolated from bovine intestine. This has much more practical significance than bacterial alkaline phosphatase, since it can be readily inactivated from the reaction mixture using proteinase K or by heating at 650C for 30 minutes or 750C for 15 minutes in the presence of 10mM EGTA.
c) Shrimp alkaline phosphatase Extracted from cold water shrimp, can be inactivated readily by heating at 650C for 15 min.
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