[Skip to Content](https://www.sigmaaldrich.com#main-content) [![Merck](https://www.sigmaaldrich.com/static/logos/purple/merck.svg)](https://www.sigmaaldrich.com/CZ/en) Products Cart0 CZEN Products ProductsApplicationsServicesResourcesSupport [Login / Register](https://www.sigmaaldrich.com/oidc-sign-in) [Order Lookup](https://www.sigmaaldrich.com/CZ/en/order-lookup) [Quick Order](https://www.sigmaaldrich.com/CZ/en/quick-order) Cart0 [Home](https://www.sigmaaldrich.com/CZ/en)[Cloning & Expression](https://www.sigmaaldrich.com/CZ/en/applications/genomics/cloning-and-expression)Dephosphorylation Procedures for DNA and Proteins # 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 ProcedureRecommended Product [1. Dephosphorylation of DNA using Calf Intestinal Alkaline Phosphatase](https://www.sigmaaldrich.com#bovine)[P4978](https://www.sigmaaldrich.com/CZ/en/product/sigma/p4978) [2. Dephosphorylation of DNA using Shrimp Alkaline Phosphatase](https://www.sigmaaldrich.com#shrimp)[P9088](https://www.sigmaaldrich.com/CZ/en/product/sigma/p9088) [3. Dephosphorylation of Protein using Bovine Intestinal Alkaline Phosphatase](https://www.sigmaaldrich.com#protein)[P0114](https://www.sigmaaldrich.com/CZ/en/product/sigma/p0114) ### [](https://www.sigmaaldrich.com)1. Dephosphorylation of DNA using Calf Intestinal Alkaline Phosphatase __Materials__ Bovine Intestinal Alkaline Phosphatase (Product No. [P4978](https://www.sigmaaldrich.com/CZ/en/product/sigma/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](https://www.sigmaaldrich.com/CZ/en/product/sigma/P3803) or [P2069](https://www.sigmaaldrich.com/CZ/en/product/sigma/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. [https://doi.org/10.1515/bchm2.1980.361.1.543](https://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. ### [](https://www.sigmaaldrich.com)2. Dephosphorylation of DNA Using Shrimp Alkaline Phosphatase Shrimp Alkaline Phosphatase (Product No. [A2237](https://www.sigmaaldrich.com/CZ/en/product/sigma/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. [https://doi.org/10.1016/0305-0491(91)90139-5](https://doi.org/10.1016/0305-0491%2891%2990139-5) 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. [https://doi.org/10.1016/s0022-2836(67)80022-6](https://doi.org/10.1016/s0022-2836%2867%2980022-6) ### [](https://www.sigmaaldrich.com)3. Dephosphorylation of Protein using Bovine Intestinal Alkaline Phosphatase __Procedure__: Incubate 100 units of alkaline phosphatase (Product No. [P0114](https://www.sigmaaldrich.com/CZ/en/product/sigma/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. [https://doi.org/10.1161/01.cir.102.11.1221](https://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. [https://doi.org/10.1161/01.cir.102.11.1221](https://doi.org/10.1161/01.cir.102.11.1221) __Related Protocols__ - [Competent Cell Protocols](https://www.sigmaaldrich.com/CZ/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/microbial-cell-culture/competent-cells) - [Calcium Phosphate Transfection Kit Protocol](https://www.sigmaaldrich.com/CZ/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/transfection-and-gene-editing/calcium-phosphate-transfection-kit) - [Escort™ III Transfection Reagent Protocol](https://www.sigmaaldrich.com/CZ/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/transfection-and-gene-editing/escort-iii-transfection-reagent-protocol) - [Universal Transfection Reagent Protocol](https://www.sigmaaldrich.com/CZ/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/transfection-and-gene-editing/universal-transfection-reagent-general) - [Protocols for Transfecting Common Cell Lines with X-tremeGENE™ Transfection Reagents](https://www.sigmaaldrich.com/CZ/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/transfection-and-gene-editing/xtg-cell-protocols) - [Co-transfection of Plasmid DNA](https://www.sigmaaldrich.com/CZ/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/transfection-and-gene-editing/xtg-cotransfect-pdna) - [X-tremeGENE™ 9 DNA Transfection Reagent Protocol](https://www.sigmaaldrich.com/CZ/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/transfection-and-gene-editing/xtg9-general-protocol) - [X-tremeGENE™ HP DNA Transfection Reagent Protocol](https://www.sigmaaldrich.com/CZ/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/transfection-and-gene-editing/xtghp-general-protocol) Top __Sign In To Continue__ To continue reading please sign in or create an account. 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