Description
Recombinant Shrimp Alkaline Phosphatase (rSAP) is a heat-labile multipurpose alkaline phosphatase that catalyzes the dephosphorylation of DNA, RNA, and nucleotides. This recombinant enzyme replaces native SAP because it is much more stable at room temperature and is
available at higher concentrations. For activity, rSAP requires magnesium (>1mM) and is tolerant to a wide variety of buffer conditions (salt, pH, etc), and thus can be added directly to many molecular biology buffers, including PCR mixtures and most restriction enzyme buffers. rSAP is completely inactivated by incubation at 65ºC for 5 minutes.
Applications
Dephosphorylation of dNTPs from PCR reaction mixtures prior to DNA sequencing (typically in a PCR Clean-up protocol1 in combination with the use of Exonuclease I (Exo I)). Dephosphorylation of DNA prior to end-labelling (using T4 Polynucleotide Kinase). Dephosphorylation of vectors (plasmids) during cloning, in order to prevent recircularization during ligation reaction (using T4 DNA Ligase).
Features
Quantity and Specifications
Specific Activity: 1U/μl. Purified from a recombinant Pichia pastoris strain harbouring the shrimp alkaline phosphatase gene from Pandalus borealis. Product consists of 1.000U of rSAP in 1ml of 25mM Tris-HCl, pH 7.6 (4ºC), containing 5mM MgCl2 and 50% glycerol, and is free of any nuclease or other enzymatic activities.
Units
One Unit (1 U) is defined as the amount of enzyme required to catalyze the release of 1μmol/min of phosphate from 6mM 4-nitrophenyl
phosphate in 0,1M glycine-NaOH pH 10,4 containing 1mM MgCl2 and 1mM ZnCl2 at 37ºC.
Storage and Shelf Life
rSAP can be stored at -20ºC for up to 2 years. Moreover, it shows excellent stability at +4ºC (at least 6 months) and at room temperature (at least 3 months). Enzyme is resistant to multiple freeze-thaw cycles.
Activity
Optimum pH range for rSAP is 7-9. Furthermore, rSAP requires magnesium (>1mM) for activity, normally present in molecular biology buffers. Additional supplements like Zinc are not required, thus rSAP can be added directly to most reaction mixtures without any adaption.
Inhibition
rSAP is inhibited by metal chelators, inorganic phosphate and phosphate analogues.
Inactivation
rSAP is completely and irreversibly inactivated by incubation at 65ºC for 5 minutes or at 75ºC for 1 minute. No further treatment is necessary
