Shrimp Alkaline Phosphatase, Recombinant (rSAP)

Applications:
1. Dephosphorylation of DNA prior to cloning.
2. PCR Clean-Up for sequencing and SNP applications.
3. Dephosphorylation of DNA prior to end-labelling using T4 PNK or OptiKinase
4. Dephosphorylation of RNA.
5. Protein dephosphorylation.

Proven Performance – the Phosphatase benchmark

• Purified from a recombinant source
• 100% heat-inactivated in 5 min at 65°C (see Fig.1)
• Significantly improved storage stability at lower temperatures (see Fig. 2) 
• Very high specific activity 
• Removes 5’-phosphates from DNA, RNA, dNTPs and proteins 
• May be added directly to restriction enzyme digests 
• No vector purification necessary  
• Requires no supplemental zinc or other additives for activity 
• Works direct in many different buffers 
• Easy treatment of unincorporated dNTPs in PCR products prior to DNA sequencing or SNP analysis 

USB Shrimp Alkaline Phosphatase, Recombinant (rSAP)
rShrimp Alkaline Phosphatase (rSAP) is a high specific activity, heat-labile alkaline phosphatase  purified from a recombinant source and originally isolated from Pandalus borealis (arctic shrimp).  rSAP is useful in many molecular biology applications such as the dephosphorylation of phosphorylated ends of DNA or RNA for subsequent use in cloning or end-labeling of probes. In cloning, dephosphorylation prevents relegation of linearized plasmid DNA. SAP may also be used to treat unincorporated dNTPs in PCR reactions to prepare templates for DNA sequencing or SNP analysis.
rShrimp Alkaline Phosphatase has approximately the same specific activity as Calf Intestinal Alkaline Phosphatase (CIAP), and like CIAP, is active in virtually all restriction enzyme reaction buffers. Unlike CIAP, rShrimp Alkaline Phosphatase is completely and irreversibly inactivated by heating reactions at 65°C for 15 min.

rShrimp Alkaline Phosphatase is particularly useful in preparing PCR products for applications involving sequencing, SNP analysis or labeling methods. Typically, excess dNTPs remaining after PCR interfere with subsequent enzymatic reactions involving DNA synthesis. rSAP dephosphorylates all of the remaining dNTPs from the PCR mixture in one easy step.

Affymetrix  is the world-wide provider of USB Shrimp Alkaline Phosphatase and we are pleased to be offering a recombinant version of our phosphatase benchmark.  Recombinant SAP eliminates the dependence on animal sourcing and offers the added benefits of increased storage stability and batch to batch consistency while still providing identical enzymatic activity and heat inactivation profiling as our native SAP.

Q. What is the difference between rSAP and the nSAP?
A. rSAP is purified from a recombinant source (yeast) while nSAP is purified directly from the native shrimp. The enzymatic activity and heat inactivation profile of recombinant is identical to nSAP.
Q. What benefits are there to switching to rSAP from nSAP?
A. rSAP has increased storage stability compared to native SAP (i.e.,  temperatures lower than 25°C)  Increased stability mitigates any activity loss for experiments with long setup times at temperatures above -20°C, while still having excellent heat inactivation at 65°C (Fig. 1). Recombinant SAP also has larger lot sizes and better batch-to-batch consistency.
Q. Validating a new enzyme is going to be a big investment of resources, why should we bother?
A. Affymetrix  will make the entire validation process as easy as possible by supplying you with a test kit containing three different rSAP lots to compare to native SAP in your assay. Technical assistance will be provided directly by our R&D group to help you throughout the entire process.
Q. Still, I don’t want to invest all the time and resources to switch to rSAP.
A. Due to a world-wide sourcing limitation, Affymetrix is transitioning from USB native SAP to USB recombinant SAP. In addition, the price of native SAP will increase, while rSAP will remain comparable.
Q. Can I get better pricing if I switch?
A. The price of native SAP will be increasing while rSAP will remain comparable to current native SAP pricing.

Properties:
Molecular Weight: Homodimer. Monomer is 55 kDa as determined by amino acid sequence.
Optimum pH: 10.4 in glycine buffer and pH 8.0 in Tris buffer.
Optimum Temperature: 37°C
Heat-Inactivation: 65°C for 15 min.
Inhibitors: 10mM DTT, 0.1% -ME
Reaction Conditions: Active in NaCl, KCl. Requires Mg²⁺ for highest activity.

Purity:
Tested for contaminating endonucleases, exonucleases and ribonucleases.

Storage Buffer:
25mM Tris-HCl (pH 7.5), 1mM MgCl₂, 50% glycerol.

Assay Conditions:
The reaction mixture contains 100mM glycine, pH 10.4, 1mM MgCl₂, 1mM ZnCl₂, 10mM p-nitrophenyl phosphate and 0.001-0.1 units of recombinant Shrimp Alkaline Phosphatase (rSAP). The change in absorbance at 405 nm is monitored (3050 μl reaction volume).

Unit Definition:
One unit is the amount of enzyme which catalyzes the hydrolysis of 1 μmol of p-nitrophenyl phosphate per min in glycine buffer (pH 10.4) at 37°C.

Concentration:
1 unit/μl

Tested User Friendly™ Functional Test:
Dephosphorylation of restriction enzyme digested plasmids (5 – 20 pmol of 5'-ends, 0.1 -0.5 units/pmol 5'-ends). Reduces religation to < 0.5% compared to the untreated control.

PROTOCOL FOR DEPHOSPHORYLATION OF NUCLEOTIDES AND DEGRADATION OF PRIMERS PRIOR TO SEQUENCING REACTIONS OR SNP ANALYSES:
Please refer to the USB ExoSAP-IT protocol, the benchmark in PCR clean-up.
The purchase of ExoSAP-IT provides a license to the methods of PCR clean up using Exonuclease I and SAP.

Functionally Tested 10X SAP Reaction Buffer (Included, PN 70103):
200mM Tris-HCl (pH 8.0), 100mM MgCl₂.
Functionally Tested SAP Dilution Buffer (1 ml included, PN 72761):
50mM Tris-HCl (pH 8.0).

References:

1. RUAN, C. C., SAMOLS, S. B. AND FULLER, C. W. (1990) Comments 17, (No.1), United States Biochemical Corporation, Cleveland, OH.
2. WERLE, E., SCNEIDER C., RENNER, M., VÖLKER, M. AND FIEHN, W. (1994) Nucleic Acids Res. 22, 4354-4355.
3. HANKE, M. AND WINK, M. (1994) BioTechniques 17, 858-860.

Storage:
Shipped on dry ice. Store at -20°C.