shrimp alkaline phosphatase  (Thermo Fisher)


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    Name:
    Shrimp Alkaline Phosphatase SAP
    Description:
    Proven Performance the Phosphatase benchmark •100 heat inactivated in 5 min at 65°C •Significantly improved storage stability at lower temperatures see Fig 1 and 2 •Very high specific activity see Fig 3 •Removes 5 phosphates from DNA RNA dNTPs and proteins •Purified from a recombinant source •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 SAP Shrimp Alkaline Phosphatase SAP is a high specific activity heat labile alkaline phosphatase purified from a recombinant source and originally isolated from Pandalus borealis arctic shrimp SAP 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 Shrimp 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 Shrimp Alkaline Phosphatase is completely and irreversibly inactivated by heating reactions at 65°C for 15 min Shrimp 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 SAP dephosphorylates all of the remaining dNTPs from the PCR mixture in one easy step 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 providing exceptional enzymatic activity and 100 heat inactivation 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 β ME Reaction Conditions Active in NaCl KCl Requires Mg2 for highest activity Source Recombinant 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 Shrimp Alkaline Phosphatase SAP 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 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 BioTechniques17 858 860
    Catalog Number:
    783901000un
    Price:
    None
    Applications:
    PCR|PCR & Real-Time PCR|Sequencing
    Category:
    Proteins Enzymes Peptides
    Buy from Supplier


    Structured Review

    Thermo Fisher shrimp alkaline phosphatase
    Proven Performance the Phosphatase benchmark •100 heat inactivated in 5 min at 65°C •Significantly improved storage stability at lower temperatures see Fig 1 and 2 •Very high specific activity see Fig 3 •Removes 5 phosphates from DNA RNA dNTPs and proteins •Purified from a recombinant source •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 SAP Shrimp Alkaline Phosphatase SAP is a high specific activity heat labile alkaline phosphatase purified from a recombinant source and originally isolated from Pandalus borealis arctic shrimp SAP 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 Shrimp 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 Shrimp Alkaline Phosphatase is completely and irreversibly inactivated by heating reactions at 65°C for 15 min Shrimp 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 SAP dephosphorylates all of the remaining dNTPs from the PCR mixture in one easy step 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 providing exceptional enzymatic activity and 100 heat inactivation 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 β ME Reaction Conditions Active in NaCl KCl Requires Mg2 for highest activity Source Recombinant 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 Shrimp Alkaline Phosphatase SAP 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 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 BioTechniques17 858 860
    https://www.bioz.com/result/shrimp alkaline phosphatase/product/Thermo Fisher
    Average 99 stars, based on 426 article reviews
    Price from $9.99 to $1999.99
    shrimp alkaline phosphatase - by Bioz Stars, 2020-09
    99/100 stars

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    Amplification:

    Article Title: Novel single nucleotide polymorphisms in the superoxide dismutase 1 and 2 genes among children with myelomeningocele
    Article Snippet: .. The amplified exon DNA was then treated with T7 exonuclease and shrimp alkaline phosphatase (United States Biochemicals, Affymetrix, Cleveland, OH) to remove excess primers and nucleotides. .. The digested product was subjected to Sanger sequencing using the BigDyeTerminator (Applied Biosystems, Foster City, CA) reagents with nested sequencing primers and resolved on the ABI3100 Genetic Analyzer (Life Technologies Inc, Grand Island, NY).

    Purification:

    Article Title: Mutations in the 3c and 7b genes of feline coronavirus in spontaneously affected FIP cats.
    Article Snippet: .. PCR products were purified using exonuclease I and shrimp alkaline phosphatase (Fermentas) during a 30 minute incubation step in a Multi Cycler PTC 200 according to the manufacturer's instructions. ..

    Polymerase Chain Reaction:

    Article Title: Development of a Respiratory Virus Panel Test for Detection of Twenty Human Respiratory Viruses by Use of Multiplex PCR and a Fluid Microbead-Based Assay ▿
    Article Snippet: .. Following PCR, the remaining dNTPs and primers were removed by incubating the entire 25-μl PCR mixture with 2.5 U shrimp alkaline phosphatase and 10 U exonuclease (Invitrogen) for 30 min at 37°C, followed by 30 s at 99°C. .. A multiplex target-specific primer extension (TSPE) reaction was used to detect specific viral sequences amplified by RT-PCR.

    Article Title: NDUFS6 mutations are a novel cause of lethal neonatal mitochondrial complex I deficiency
    Article Snippet: .. Automated sequencing for NDUFS6 was performed on shrimp alkaline phosphatase–treated PCR products (ExoSAP-IT; USB Corp.) using a DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences). .. The sequencing reactions were purified using AutoSeq G-50 columns (Amersham Biosciences).

    Article Title: Mutations in the 3c and 7b genes of feline coronavirus in spontaneously affected FIP cats.
    Article Snippet: .. PCR products were purified using exonuclease I and shrimp alkaline phosphatase (Fermentas) during a 30 minute incubation step in a Multi Cycler PTC 200 according to the manufacturer's instructions. ..

    Incubation:

    Article Title: Mutations in the 3c and 7b genes of feline coronavirus in spontaneously affected FIP cats.
    Article Snippet: .. PCR products were purified using exonuclease I and shrimp alkaline phosphatase (Fermentas) during a 30 minute incubation step in a Multi Cycler PTC 200 according to the manufacturer's instructions. ..

    De-Phosphorylation Assay:

    Article Title: Phosphorylation Dependence and Stoichiometry of the Complex Formed by Tyrosine Hydroxylase and 14-3-3γ *
    Article Snippet: .. TH (5 μM) was then preincubated for 10 min on ice with or without 14-3-3γ (7.5 μM) prior to 5-fold dilution in dephosphorylation assay using shrimp alkaline phosphatase (SAP) (0.14 U/μl, Thermo Fisher Scientific), 25 mM Hepes, pH 7.2, 130 mM KCl, 2 mM MgCl2 , 1 mM DTT, at 15 °C or 25 °C. .. Aliquots were taken at different time points, spotted on phospho-cellulose paper, washed, and counted to measure remaining labeled protein.

    Sequencing:

    Article Title: NDUFS6 mutations are a novel cause of lethal neonatal mitochondrial complex I deficiency
    Article Snippet: .. Automated sequencing for NDUFS6 was performed on shrimp alkaline phosphatase–treated PCR products (ExoSAP-IT; USB Corp.) using a DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences). .. The sequencing reactions were purified using AutoSeq G-50 columns (Amersham Biosciences).

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    Thermo Fisher shrimp alkaline phosphatase sap
    Effect of <t>14-3-3γ</t> on TH phosphorylation and dephosphorylation. A , binding of TH phosphorylated on Ser19 by PRAK to immobilized 14-3-3γ. TH was phosphorylated to a stoichiometry of 0.17 (red) or 1.0 (blue) before preparation for injections at subunit concentrations of 5, 25, and 50 nM. Sensorgrams were scaled for illustration of kinetics. B , TH was [ 32 P]-labeled on Ser19 to different stoichiometries using PRAK before incubation with the PRAK inhibitor epigallocatechin gallate and 14-3-3γ (7.5 μM, TH-pS19:14-3-3 mixing ratio of 1:1.5). The complex was then diluted 1/10 in buffer containing high levels of shrimp alkaline phosphatase <t>(SAP)</t> (145 U/ml), and the temporal decay of 32 P-Ser19 was monitored as described under “Experimental Procedures.” Controls without 14-3-3 and without SAP were also measured (dotted lines). Exponential decay functions were fitted to each curve, and the corresponding rate constants were 0.089, 0.125, and 0.157 min −1 for TH-pS19 phosphorylated to 51% (○), 33% (▵), and 18.5% (□), respectively. Insignificant change in phosphorylation stoichiometry was observed in the absence of SAP (horizontal dotted lines), and a high rate of dephosphorylation was measured in the absence of 14-3-3γ (lower dotted lines). C , the phosphorylation of TH-pS19 (2.5 μM, pre-phosphorylated on Ser19 by PRAK) by PKA in the absence (○) or presence (●) of 14-3-3γ (10 μM).
    Shrimp Alkaline Phosphatase Sap, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 440 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/shrimp alkaline phosphatase sap/product/Thermo Fisher
    Average 99 stars, based on 440 article reviews
    Price from $9.99 to $1999.99
    shrimp alkaline phosphatase sap - by Bioz Stars, 2020-09
    99/100 stars
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    92
    Thermo Fisher exo sap
    Effect of <t>14-3-3γ</t> on TH phosphorylation and dephosphorylation. A , binding of TH phosphorylated on Ser19 by PRAK to immobilized 14-3-3γ. TH was phosphorylated to a stoichiometry of 0.17 (red) or 1.0 (blue) before preparation for injections at subunit concentrations of 5, 25, and 50 nM. Sensorgrams were scaled for illustration of kinetics. B , TH was [ 32 P]-labeled on Ser19 to different stoichiometries using PRAK before incubation with the PRAK inhibitor epigallocatechin gallate and 14-3-3γ (7.5 μM, TH-pS19:14-3-3 mixing ratio of 1:1.5). The complex was then diluted 1/10 in buffer containing high levels of shrimp alkaline phosphatase <t>(SAP)</t> (145 U/ml), and the temporal decay of 32 P-Ser19 was monitored as described under “Experimental Procedures.” Controls without 14-3-3 and without SAP were also measured (dotted lines). Exponential decay functions were fitted to each curve, and the corresponding rate constants were 0.089, 0.125, and 0.157 min −1 for TH-pS19 phosphorylated to 51% (○), 33% (▵), and 18.5% (□), respectively. Insignificant change in phosphorylation stoichiometry was observed in the absence of SAP (horizontal dotted lines), and a high rate of dephosphorylation was measured in the absence of 14-3-3γ (lower dotted lines). C , the phosphorylation of TH-pS19 (2.5 μM, pre-phosphorylated on Ser19 by PRAK) by PKA in the absence (○) or presence (●) of 14-3-3γ (10 μM).
    Exo Sap, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exo sap/product/Thermo Fisher
    Average 92 stars, based on 96 article reviews
    Price from $9.99 to $1999.99
    exo sap - by Bioz Stars, 2020-09
    92/100 stars
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    94
    Thermo Fisher enzymatic mixture exo sap
    Effect of <t>14-3-3γ</t> on TH phosphorylation and dephosphorylation. A , binding of TH phosphorylated on Ser19 by PRAK to immobilized 14-3-3γ. TH was phosphorylated to a stoichiometry of 0.17 (red) or 1.0 (blue) before preparation for injections at subunit concentrations of 5, 25, and 50 nM. Sensorgrams were scaled for illustration of kinetics. B , TH was [ 32 P]-labeled on Ser19 to different stoichiometries using PRAK before incubation with the PRAK inhibitor epigallocatechin gallate and 14-3-3γ (7.5 μM, TH-pS19:14-3-3 mixing ratio of 1:1.5). The complex was then diluted 1/10 in buffer containing high levels of shrimp alkaline phosphatase <t>(SAP)</t> (145 U/ml), and the temporal decay of 32 P-Ser19 was monitored as described under “Experimental Procedures.” Controls without 14-3-3 and without SAP were also measured (dotted lines). Exponential decay functions were fitted to each curve, and the corresponding rate constants were 0.089, 0.125, and 0.157 min −1 for TH-pS19 phosphorylated to 51% (○), 33% (▵), and 18.5% (□), respectively. Insignificant change in phosphorylation stoichiometry was observed in the absence of SAP (horizontal dotted lines), and a high rate of dephosphorylation was measured in the absence of 14-3-3γ (lower dotted lines). C , the phosphorylation of TH-pS19 (2.5 μM, pre-phosphorylated on Ser19 by PRAK) by PKA in the absence (○) or presence (●) of 14-3-3γ (10 μM).
    Enzymatic Mixture Exo Sap, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/enzymatic mixture exo sap/product/Thermo Fisher
    Average 94 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    enzymatic mixture exo sap - by Bioz Stars, 2020-09
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    Effect of 14-3-3γ on TH phosphorylation and dephosphorylation. A , binding of TH phosphorylated on Ser19 by PRAK to immobilized 14-3-3γ. TH was phosphorylated to a stoichiometry of 0.17 (red) or 1.0 (blue) before preparation for injections at subunit concentrations of 5, 25, and 50 nM. Sensorgrams were scaled for illustration of kinetics. B , TH was [ 32 P]-labeled on Ser19 to different stoichiometries using PRAK before incubation with the PRAK inhibitor epigallocatechin gallate and 14-3-3γ (7.5 μM, TH-pS19:14-3-3 mixing ratio of 1:1.5). The complex was then diluted 1/10 in buffer containing high levels of shrimp alkaline phosphatase (SAP) (145 U/ml), and the temporal decay of 32 P-Ser19 was monitored as described under “Experimental Procedures.” Controls without 14-3-3 and without SAP were also measured (dotted lines). Exponential decay functions were fitted to each curve, and the corresponding rate constants were 0.089, 0.125, and 0.157 min −1 for TH-pS19 phosphorylated to 51% (○), 33% (▵), and 18.5% (□), respectively. Insignificant change in phosphorylation stoichiometry was observed in the absence of SAP (horizontal dotted lines), and a high rate of dephosphorylation was measured in the absence of 14-3-3γ (lower dotted lines). C , the phosphorylation of TH-pS19 (2.5 μM, pre-phosphorylated on Ser19 by PRAK) by PKA in the absence (○) or presence (●) of 14-3-3γ (10 μM).

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Phosphorylation Dependence and Stoichiometry of the Complex Formed by Tyrosine Hydroxylase and 14-3-3γ *

    doi: 10.1074/mcp.M113.035709

    Figure Lengend Snippet: Effect of 14-3-3γ on TH phosphorylation and dephosphorylation. A , binding of TH phosphorylated on Ser19 by PRAK to immobilized 14-3-3γ. TH was phosphorylated to a stoichiometry of 0.17 (red) or 1.0 (blue) before preparation for injections at subunit concentrations of 5, 25, and 50 nM. Sensorgrams were scaled for illustration of kinetics. B , TH was [ 32 P]-labeled on Ser19 to different stoichiometries using PRAK before incubation with the PRAK inhibitor epigallocatechin gallate and 14-3-3γ (7.5 μM, TH-pS19:14-3-3 mixing ratio of 1:1.5). The complex was then diluted 1/10 in buffer containing high levels of shrimp alkaline phosphatase (SAP) (145 U/ml), and the temporal decay of 32 P-Ser19 was monitored as described under “Experimental Procedures.” Controls without 14-3-3 and without SAP were also measured (dotted lines). Exponential decay functions were fitted to each curve, and the corresponding rate constants were 0.089, 0.125, and 0.157 min −1 for TH-pS19 phosphorylated to 51% (○), 33% (▵), and 18.5% (□), respectively. Insignificant change in phosphorylation stoichiometry was observed in the absence of SAP (horizontal dotted lines), and a high rate of dephosphorylation was measured in the absence of 14-3-3γ (lower dotted lines). C , the phosphorylation of TH-pS19 (2.5 μM, pre-phosphorylated on Ser19 by PRAK) by PKA in the absence (○) or presence (●) of 14-3-3γ (10 μM).

    Article Snippet: TH (5 μM) was then preincubated for 10 min on ice with or without 14-3-3γ (7.5 μM) prior to 5-fold dilution in dephosphorylation assay using shrimp alkaline phosphatase (SAP) (0.14 U/μl, Thermo Fisher Scientific), 25 mM Hepes, pH 7.2, 130 mM KCl, 2 mM MgCl2 , 1 mM DTT, at 15 °C or 25 °C.

    Techniques: De-Phosphorylation Assay, Binding Assay, Labeling, Incubation