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Dephosphorylation Procedures for DNA and Proteins

Dephosphorylation of Proteins and DNA

Dephosphorylation is the removal of a phosphate (PO43−) group by hydrolysis. To dephosphorylate a protein or DNA, an enzyme or hydrolase that cleaves ester bonds is required. For example, phosphatases remove phosphate groups by hydrolyzing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl (−OH) group.

Procedures for Dephosphorylation

1. Dephosphorylation of DNA using Calf Intestinal Alkaline Phosphatase

Materials

Bovine Intestinal Alkaline Phosphatase (Product No. P4978)
10X CIP Buffer (Product No. C3225)
1 M NaCl
0.5 M Tris-HCl pH 7.9 at 25 °C
0.1 M MgCl2
0.01 M dithiothreitol

Storage buffer

10 mM Tris-HCl, pH 8.2
50 mM KCl
1 mM MgCl2
0.1 mM ZnCl2
50% glycerol

Procedure:

  1. Dissolve DNA in 1X CIP Buffer (0.5 µg DNA/10 µL).
  2. For 5’ overhang DNA add 0.1 units/pmol CIP; for 3’ overhang or blunt end DNA add 1 unit/pmol.
  3. Incubate 60 minutes at 37 °C.
  4. Extract with phenol/chloroform2 (Product No. P3803 or P2069) or gel purify the DNA.*
  5. 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.

References

1.
MÖSSNER E, BOLL M, PFLEIDERER G. 1980. Purification of Human and Bovine Alkaline Phosphatases by Affinity Chromatography. Hoppe-Seyler´s Zeitschrift für physiologische Chemie. 361(1):543-550. http://dx.doi.org/10.1515/bchm2.1980.361.1.543
2.
Sambrook Jea. 1989. Molecular Cloning: A Laboratory Manual. p 5.72, 6.22-6.47 and E.3-E.13.. Cold Spring Harbor Laboratory.

2. Dephosphorylation of DNA Using Shrimp Alkaline Phosphatase

Shrimp Alkaline Phosphatase (Product No. A2237) 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.

Perform dephosphorylation 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.

References

1.
Olsen RL, Øverbø K, Myrnes B. 1991. Alkaline phophatase from the hepatopancreas of shrimp (Pandalus borealis): A dimeric enzyme with catalytically active subunits. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry. 99(4):755-761. http://dx.doi.org/10.1016/0305-0491(91)90139-5
2.
Sambrook J, Russell D. 2001. Molecular Cloning: A Laboratory Manual. 3. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
3.
Takanami M. 1967. Analysis of the 5?-terminal nucleotide sequences of ribonucleic acids. Journal of Molecular Biology. 23(2):135-148. http://dx.doi.org/10.1016/s0022-2836(67)80022-6

3. Dephosphorylation of Protein using Bovine Intestinal Alkaline Phosphatase

Procedure: Incubate 100 units of alkaline phosphatase (Product No. P0114) with 400 µg 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.

1.
Labugger R, Organ L, Collier C, Atar D, Van Eyk JE. 2000. Extensive Troponin I and T Modification Detected in Serum From Patients With Acute Myocardial Infarction. Circulation. 102(11):1221-1226. http://dx.doi.org/10.1161/01.cir.102.11.1221

References

1.
Labugger R, Organ L, Collier C, Atar D, Van Eyk JE. 2000. Extensive Troponin I and T Modification Detected in Serum From Patients With Acute Myocardial Infarction. Circulation. 102(11):1221-1226. http://dx.doi.org/10.1161/01.cir.102.11.1221