Procedures for Dephosphorylation
1. Dephosphorylation of DNA using Calf Intestinal Alkaline Phosphatase
Bovine Intestinal Alkaline Phosphatase, Cat. No. P4978
10X CIP Buffer (Cat. No. C3225)
1 M NaCl
0.5 M Tris-HCl pH 7.9 at 25 °C
0.1 M MgCl2
0.01 M dithiothreitol
10 mM Tris-HCl, pH 8.2
50 mM KCl
1 mM MgCl2
0.1 mM ZnCl2
- Dissolve DNA in 1X CIP Buffer (0.5 µg DNA/10 µl).
- For 5’ overhang DNA add 0.1 units/pmol CIP; for 3’ overhang or blunt end DNA add 1 unit/pmol.
- Incubate 60 minutes at 37 °C.
- Extract with phenol/chloroform2 (P3803 or P2069) or gel purify the DNA.*
- Recover the DNA by alcohol precipitation.2
*Note: Phenol extraction or gel purification makes heat inactivation unnecessary.
Heat Inactivation: For the bovine intestinal enzyme, greater than 95% of the activity can be inactivated by heating to 75 °C for 10 minutes in the presence of 5 mM EDTA. Note: Alkaline phosphatase from E. coli is more heat stable than the bovine intestinal enzyme and is more resistant to heat inactivation.
- Moessner,E. et al., Z. Physiol. Chem. 361, 543.
- Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989), p 5.72, 6.22-6.47 and E.3-E.13.
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2. Dephosphorylation of DNA using Shrimp Alkaline Phosphatase
Shrimp Alkaline Phosphatase, Cat. No. P9088 is supplied as a solution in 50% glycerol containing 25 mM Tris-HCl, pH 7.6, 1 mM MgCl2 and 0.1 mM ZnCl2.
Dilutions can be prepared in 0.05 M Tris-HCl, pH 8.5 containing 5 mM MgCl2.
Dephosphorylation should be carried out in 0.05 M Tris-HCl, 5 mM MgCl2, pH 8.5.
Heat Inactivation: After the dephosphorylation reaction, the enzyme can then be inactivated by warming to 60 °C for 15 minutes.
- Olsen, R., Overbo, K., Myrnes, B., Alkaline phosphatase from the hepatopancreas of shrimp (Pandalus borealis): a dimeric enzyme with catalytically active subunits, Comp. Biochem. Physiol., 99B, 755-761,1991.
- Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
- Takanami, M., Analysis of the 5'-terminal nucleotide sequences of ribonucleic acids 1. the 5'-termini of Escherichia coli ribosomal RNA J. Mol. Biol. 23, 135 (1967).
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3. Dephosphorylation of Protein using Bovine Intestinal Alkaline Phosphatase
- Incubate 100 units of alkaline phosphatase, Cat. No. P0114 with 400 ug of protein in 5 mM Tris pH 7.9, 10 mM NaCl, 1 mM MgCl2, and 0.1 mM DTT for 30 minutes at 30 °C.
- For SDS PAGE analysis, the reaction can be stopped with the addition of 5X sample buffer and boiling.
- Labugger R, et al. Extensive Troponin I and T Modification Detected in Serum from Patients with Acute Myocardial Infarction. Circulation 102(11): 1221-1226 (2000).
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