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Phosphatases and Kinase

Shrimp Alkaline Phosphatase (SAP)

Product information

Features

• 100% active in restriction enzyme, PCR and RT buffers.
• Completely inactive after 15 min incubation at 65°C.

Applications

• Dephosphorylation of termini:
  – DNA and RNA (1),
  – cloning vector DNA to avoid recircularization,
  – DNA and RNA 5'-ends prior to labeling with T4 polynucleotide kinase.
• Degradation of dNTPs in PCR mixture prior to sequencing of PCR product (2).
• Dephosphorylation of proteins (3).

Description

Shrimp Alkaline Phosphatase (SAP) catalyzes the release of 5'- and 3'-phosphate groups from DNA, RNA and nucleotides. Also this enzyme can remove phosphate groups from proteins.

Source

Arctic shrimp Pandalus borealis.

Molecular Weight

This enzyme is homodimer. It consists of two identical subunits of 54 kDa (4).

Definition of Activity Unit

One unit of the enzyme hydrolyzes 1 µmol of 4-nitrophenylphosphate in 1 min at 37°C.

Enzyme activity is assayed in the following mixture: 1 M diethanolamine-HCl (pH 9.8), 0.5 mM MgCl₂, 10 mM 4-nitrophenylphosphate.

Storage Buffer

The enzyme is supplied in:
25 mM Tris-HCl (pH 7.6 at 4°C), 1 mM MgCl₂, 0.1 mM ZnCl₂ and 50% (v/v) glycerol.

10X Reaction Buffer

0.1 M Tris-HCl (pH 7.5 at 37°C), 0.1 M MgCl₂ and 1 mg/ml BSA.

Quality Control

The absence of endo-, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests. Functionally tested for dephosphorylation of DNA 5'-termini.

Inhibition and Inactivation

• Inhibitors: metal chelators, inorganic phosphate and phosphate analogs.
• Inactivated by heating at 65°C for 15 min.

Note

Binding of SAP to DNA may result in a band shift in agarose gels. To avoid this, incubate samples with 6X DNA Loading Dye & SDS Solution at 65°C for 10 min and chill on ice prior to electrophoresis.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Dephosphorylation of DNA 5'-termini with Shrimp Alkaline Phosphatase

This protocol is suitable for removal of 3'- and 5'-phosphate groups from DNA and RNA. The protocol below is an example for dephosphorylation of ~3 kb linear vector DNA.

1. Prepare the following reaction mixture:
   

   Linear DNA (~3 kb plasmid)	1 µg (~1 pmol termini)
   10X SAP reaction buffer	2 µl
   Shrimp Alkaline Phosphatase (SAP)	1 µl (1 u)
   Water, nuclease-free	to 20 µl
   Total volume	20 µl

2. Mix thoroughly, spin briefly and incubate at 37°C for 30 min (5'-overhangs or blunt ends) or 60 min (3'-overhangs).
3. Stop reaction by heating at 65°C for 15 min.

Note

For efficient dephosphorylation plasmid DNA should be free of RNA and genomic DNA.

PCR Product Clean-Up Prior to Sequencing

The clean-up reaction removes unincorporated primers and degrades unincorporated nucleotides. The resulting PCR product is ready to use for sequencing without additional purification, e.g. using column purification kits.

1. Prepare the following reaction mixture:
   

   PCR mixture (directly after completion of PCR)	5 µl
   Exonuclease I (Exo I)	0.5 µl (10 u)
   FastAP™ Thermosensitive Alkaline Phosphatase or Shrimp Alkaline Phosphatase (SAP)	1 µl (1 u)

2. Mix well and incubate at 37°C for 15 min.
3. Stop the reaction by heating the mixture at 85°C for 15 min.

Note

• Up to 5 µl of purified PCR products can be used directly for DNA sequencing without further purification.
• For reliable sequencing results there should not be non-specific PCR products.
• The protocol may be applied for clean-up of PCR products, generated by any thermophilic DNA polymerase or polymerase mix.
• The procedure is not recommended for downstream cloning applications.

Reference

1. Werle, E., et al., Convenient single-step, one tube purification of PCR products for direct sequencing, Nucleic Acids Res., 22, 4354-4355, 1994.

Radioactive Labeling of RiboRuler™ RNA Ladders by T4 Polynucleotide Kinase

The ready-to-use versions of RiboRuler™ RNA ladders can not be radiolabeled with T4 Polynucleotide Kinase.
For efficient labeling of RNA ladders it is recommended to remove 5'-phosphate groups from RNA and then phosphorylate in forward reaction using T4 Polynucleotide Kinase.
I. Dephosphorylation

1. Prepare the following reaction mixture:

   RiboRuler™ Low Range RNA Ladder or RiboRuler™ High Range RNA Ladder	8 µl
   RiboLock™ RNase Inhibitor	0.5 µl (20 u)
   10X reaction buffer for alkaline phosphatase	2 µl
   FastAP™ Thermosensitive Alkaline Phosphatase or Shrimp Alkaline Phosphatase	2 µl (2 u)
   DEPC-treated Water	to 20 µl
   Total volume	20 µl

2. Incubate at 37°C for 30 minutes.
3. Remove proteins from the mixture with a 20 µl aliquot of Tris-saturated (pH 8.0) phenol and chloroform mixture. Save the upper aqueous phase and extract it twice with 20 µl aliquot of chloroform.
4. Precipitate RNA by adding 1 µl of 3 M Sodium Acetate Solution, 55 µl of 96% ethanol and keep 15-30 min at -20°C. Centrifuge the mixture for 20 min at 10,000-15,000 rpm at 4°C.
5. Rinse the pellet with 20 µl cold 75% ethanol. Centrifuge 10 min at 10,000-15,000 rpm, 4°C.
6. Discard the supernatant and dissolve the air-dried pellet in 10 µl of DEPC-treated Water.

II. Labeling

1. Prepare the following reaction mixture:

   Dephosphorylated RiboRuler™ Low Range RNA Ladder or Dephosphorylated RiboRuler™ High Range RNA Ladder	1 µl 2.5 µl
   [gamma-32P]-ATP (5000 Ci/mmol,10 µCi/µl)*	5 µl (10 pmol)
   RiboLock™ RNase Inhibitor	0.25 µl (10 u)
   10X buffer A for forward reaction (supplied with T4 polynucleotide kinase)	1 µl
   T4 Polynucleotide Kinase	1 µl (10 u)
   DEPC-treated Water	to 10 µl
   Total volume	10 µl

   * If [gamma-³²P]-ATP with a high specific activity (higher than 5000 Ci/mmol) is used, the label can be diluted with ATP. Total ATP concentration should be at least 1 µM.
2. Incubate at 37°C for 30 minutes.
3. Stop the reaction by adding 1 µl of 0.5 M EDTA, pH 8.0 and extract the mixture with an equal volume of chloroform.
4. Determine the efficiency of label incorporation.
5. Load the ladder on the gel.

Dephosphorylation of Proteins

Reaction Mixture:
1X FastAP™ reaction buffer,
0.1-0.2 mg/ml of phosphoprotein,
10 u of FastAP™ Thermosensitive Alkaline Phosphatase.
Incubate at 37°C for 1 hour.

Note

• The reaction can be stopped by addition of a final concentration of 50 mM EDTA (#R1021) or by addition of a final concentration of 10 mM sodium orthovanadate (Na₃VO₄).
• The optimal incubation time and the enzyme concentration must be determined experimentally for each substrate.

References

1. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
2. Werle, E., et al., Convenient single-step, one tube purification of PCR products for direct sequencing, Nucleic Acids Res., 22, 4354-4355, 1994.
3. Khosravi, R., et al., Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage, Proc. Natl. Acad. Sci USA, 96, 14973-14977, 1999.
4. Nilsen, I.W., et al., Thermolabile alkaline phosphatase from Northern shrimp (Padalus borealis): protein and cDNA sequence analyses, Comparative Biochemistry and Physiology, B, 129, 853-861, 2001.

Related products

• T4 Polynucleotide Kinase (T4 PNK)
• Exonuclease I (Exo I)
• T4 DNA Ligase
• T4 RNA Ligase
• Rapid DNA Ligation Kit
• CloneJET™ PCR Cloning Kit
• InsTAclone™ PCR Cloning Kit
• 6X DNA Loading Dye & SDS Solution
• Water, nuclease-free