calf intestinal alkaline phosphatase  (New England Biolabs)


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    Name:
    Alkaline Phosphatase Calf Intest CIP
    Description:
    Alkaline Phosphatase Calf Intest CIP 5 000 units
    Catalog Number:
    M0290L
    Price:
    342
    Size:
    5 000 units
    Category:
    Alkaline Phosphatases
    Score:
    85
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    Structured Review

    New England Biolabs calf intestinal alkaline phosphatase
    Alkaline Phosphatase Calf Intest CIP
    Alkaline Phosphatase Calf Intest CIP 5 000 units
    https://www.bioz.com/result/calf intestinal alkaline phosphatase/product/New England Biolabs
    Average 99 stars, based on 141 article reviews
    Price from $9.99 to $1999.99
    calf intestinal alkaline phosphatase - by Bioz Stars, 2019-10
    99/100 stars

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    Clone Assay:

    Article Title: Long-Chain N-Acyl Amino Acid Synthases Are Linked to the Putative PEP-CTERM/Exosortase Protein-Sorting System in Gram-Negative Bacteria
    Article Snippet: PCRs were carried out under the following conditions using a Biometra TGradient thermocycler: 100-μl reaction mixtures consisted of 1.25× Thermopol buffer (NEB), 500 μM deoxynucleoside triphosphate (dNTP) mix, primers at 0.4 μM each, 100 ng of template DNA, and 0.5 μl per reaction of both Phusion Hot Start DNA Polymerase and Dynazyme II DNA Polymerase (NEB-Finnzymes); the PCR program consisted of 1 cycle at 95°C for 120 s, followed by 31 cycles of 30 s at 97°C, 30 s at 64°C, and 50 s at 72°C, followed by 120 s at 72°C. .. All PCR amplicons were subsequently digested with NdeI and PstI (except for the ExoATC3 gene amplicon, which required NcoI and SbfI) in preparation for cloning into the custom E. coli expression vector p4R-Ptac (Epoch Biolabs, Inc., Sugarland, TX), which was prepared by reciprocal enzymatic digestion and calf intestinal phosphatase (CIP) treatment (NEB). .. Insert and vector samples were ligated (Fast-Link DNA Ligation kit; Epicentre), transformed into Transformax electrocompetent E. coli EC100 (Epicentre), and selected on Luria-Bertani agar (LB-agar) containing 200 μg ml−1 ampicillin.

    Article Title: Deletion of v-chiA from a Baculovirus Reduces Horizontal Transmission in the Field
    Article Snippet: Paragraph title: Cloning, sequence analysis, and generation of recombinant viruses. ... The 12.1-kbp band from isolate 203-WT and the 8.3-kbp band from 203-NL were excised from a Tris-borate-EDTA (TBE) agarose gel, purified from the agarose using the GeneClean spin kit (Bio101), and ligated into SpeI-digested pBluescriptSK+ vector after dephosphorylation using calf intestinal alkaline phosphatase (New England BioLabs).

    Article Title: Mutants Resistant to LpxC Inhibitors by Rebalancing Cellular Homeostasis
    Article Snippet: The vector was treated with calf intestinal alkaline phosphatase (New England Biolabs). .. Confirmed constructs were transformed into chemically competent W3110 as described previously ( ).

    Article Title: Requirement of the galU Gene for Polysaccharide Production by and Pathogenicity and Growth In Planta of Xanthomonas citri subsp. citri
    Article Snippet: The resulting 1.9-kb fragment was ligated to PCR 2.1-TOPO using the manufacturer's protocol (Invitrogen, Carlsbad, CA), resulting in pCGU1.1. .. A BamHI fragment containing the galU gene was isolated from pCGU1.1 and cloned into similarly digested pUFR053 that was treated with calf intestinal alkaline phosphatase (New England Biolabs, Ipswich, MA) , resulting in pCGU2.1 (Table ). .. Plasmid pCGU2.1 was transferred into galU mutants D12 and F6 ( galU ::Tn 5 ) by triparental mating with an E. coli helper strain containing pRK2013 ( ).

    Article Title: The RAS-Binding Domain of Human BRAF Protein Serine/Threonine Kinase Exhibits Allosteric Conformational Changes upon Binding HRAS
    Article Snippet: Paragraph title: Cloning, Expression, Purification, and Sample Preparation ... To activate HRAS for binding to BRAF RBD, the purified bacterially expressed GDP form of HRAS was treated with calf intestinal alkaline phosphatase (New England BioLabs) in the presence of a non-hydrolyzable analog of GTP, GppNHp (Sigma) ( ).

    Centrifugation:

    Article Title: The RAS-Binding Domain of Human BRAF Protein Serine/Threonine Kinase Exhibits Allosteric Conformational Changes upon Binding HRAS
    Article Snippet: To activate HRAS for binding to BRAF RBD, the purified bacterially expressed GDP form of HRAS was treated with calf intestinal alkaline phosphatase (New England BioLabs) in the presence of a non-hydrolyzable analog of GTP, GppNHp (Sigma) ( ). .. Samples of [ U -13 C,15 N]- and [ U -5%-13 C,100%-15 N]-BRAF RBD for NMR structure determination were concentrated by centrifugation to 0.7–0.9 mM in 20 mM ammonium acetate, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2 , 50 µM 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS), and 10% 2 H2 O (v/v) at pH 4.5.

    Amplification:

    Article Title: Long-Chain N-Acyl Amino Acid Synthases Are Linked to the Putative PEP-CTERM/Exosortase Protein-Sorting System in Gram-Negative Bacteria
    Article Snippet: PCRs were carried out under the following conditions using a Biometra TGradient thermocycler: 100-μl reaction mixtures consisted of 1.25× Thermopol buffer (NEB), 500 μM deoxynucleoside triphosphate (dNTP) mix, primers at 0.4 μM each, 100 ng of template DNA, and 0.5 μl per reaction of both Phusion Hot Start DNA Polymerase and Dynazyme II DNA Polymerase (NEB-Finnzymes); the PCR program consisted of 1 cycle at 95°C for 120 s, followed by 31 cycles of 30 s at 97°C, 30 s at 64°C, and 50 s at 72°C, followed by 120 s at 72°C. .. All PCR amplicons were subsequently digested with NdeI and PstI (except for the ExoATC3 gene amplicon, which required NcoI and SbfI) in preparation for cloning into the custom E. coli expression vector p4R-Ptac (Epoch Biolabs, Inc., Sugarland, TX), which was prepared by reciprocal enzymatic digestion and calf intestinal phosphatase (CIP) treatment (NEB). .. Insert and vector samples were ligated (Fast-Link DNA Ligation kit; Epicentre), transformed into Transformax electrocompetent E. coli EC100 (Epicentre), and selected on Luria-Bertani agar (LB-agar) containing 200 μg ml−1 ampicillin.

    Article Title: Mutants Resistant to LpxC Inhibitors by Rebalancing Cellular Homeostasis
    Article Snippet: Wild-type fabZ was amplified using genomic DNA from W3110 and primers 7 and 8. .. The vector was treated with calf intestinal alkaline phosphatase (New England Biolabs).

    Article Title: Requirement of the galU Gene for Polysaccharide Production by and Pathogenicity and Growth In Planta of Xanthomonas citri subsp. citri
    Article Snippet: The entire galU gene with 624 bp of upstream sequence and 388 bp of downstream sequence was amplified from genomic DNA of X. citri subsp. citri wild-type strain 306 by a PCR performed with primers CGU-F and CGU-R, which contained a BamHI restriction site (Table ). .. A BamHI fragment containing the galU gene was isolated from pCGU1.1 and cloned into similarly digested pUFR053 that was treated with calf intestinal alkaline phosphatase (New England Biolabs, Ipswich, MA) , resulting in pCGU2.1 (Table ).

    Mass Spectrometry:

    Article Title: Ubp-M serine 552 phosphorylation by cyclin-dependent kinase 1 regulates cell cycle progression
    Article Snippet: For this purpose, we treated Ubp-M with Calf Intestinal Alkaline Phosphatase (CIP, NEB). .. For this purpose, we treated Ubp-M with Calf Intestinal Alkaline Phosphatase (CIP, NEB).

    Article Title: Switching of Self-Assembly in a Peptide Nanostructure with a Specific Enzyme
    Article Snippet: Following this time, calf intestinal alkaline phosphatase (CIP, New England Biolabs) was added (60 U/mg PA) and the reaction was incubated for an additional 30 minutes at 37°C. .. For control experiments involving PA 3 with PKA or PA 1 with PKB, conditions were otherwise kept identical except for these two substitutions.

    Autoradiography:

    Article Title: Transcriptome-wide microRNA and target dynamics in the fat body during the gonadotrophic cycle of Aedes aegypti
    Article Snippet: Beads were further digested with 1:1,000 RNase A dilution, dephosphorylated with calf intestinal alkaline phosphatase (CIP) (NEB) and ligated to 32 P-labeled 3′ RNA linker (RL3-OH) using T4 RNA ligase (Thermo Fisher Scientific). .. Beads were further digested with 1:1,000 RNase A dilution, dephosphorylated with calf intestinal alkaline phosphatase (CIP) (NEB) and ligated to 32 P-labeled 3′ RNA linker (RL3-OH) using T4 RNA ligase (Thermo Fisher Scientific).

    Construct:

    Article Title: Long-Chain N-Acyl Amino Acid Synthases Are Linked to the Putative PEP-CTERM/Exosortase Protein-Sorting System in Gram-Negative Bacteria
    Article Snippet: Paragraph title: Construction of heterologous expression constructs. ... All PCR amplicons were subsequently digested with NdeI and PstI (except for the ExoATC3 gene amplicon, which required NcoI and SbfI) in preparation for cloning into the custom E. coli expression vector p4R-Ptac (Epoch Biolabs, Inc., Sugarland, TX), which was prepared by reciprocal enzymatic digestion and calf intestinal phosphatase (CIP) treatment (NEB).

    Article Title: Mutants Resistant to LpxC Inhibitors by Rebalancing Cellular Homeostasis
    Article Snippet: The vector was treated with calf intestinal alkaline phosphatase (New England Biolabs). .. The vector was treated with calf intestinal alkaline phosphatase (New England Biolabs).

    Incubation:

    Article Title: Afferent Regulation of Chicken Auditory Brainstem Neurons: Rapid Changes in Phosphorylation of Elongation Factor 2
    Article Snippet: A western blot immunoassay was conducted to confirm the specificity of anti-eEF2 and anti-p-eEF2 used in the present study. .. For phosphatase treatment, HEK293 cells were incubated in a lysis buffer with Calf Intestinal Phosphatase (CIP; # M0290S; New England Biolabs, Ipswich, MA) at 1 unit CIP per 1 μg protein for 30 minutes at 37ºC before harvest. .. Brain protein samples were harvested from the NM and the surrounding region in the dorsocaudal brainstem of chicks.

    Article Title: Genome-wide assessment of sequence-intrinsic enhancer responsiveness at single-base-pair resolution
    Article Snippet: 24 h after electroporation total RNA was isolated followed by polyA+ RNA purification and DNaseI treatment, as described previously . .. 10–20 µg (focused) or 200 µg (genome-wide) of DNaseI-treated RNA was incubated with calf intestinal alkaline phosphatase (CIP; NEB cat. no. M0290L). .. Per 1 µg RNA, 0.5 µl CIP was used.

    Article Title: Switching of Self-Assembly in a Peptide Nanostructure with a Specific Enzyme
    Article Snippet: This was also supplemented with 1 mM ATP. cAMP-dependent Protein Kinase (PKA) catalytic subunit (New England Biolabs) was added to the mixture (10,000 U/mg PA). .. Following this time, calf intestinal alkaline phosphatase (CIP, New England Biolabs) was added (60 U/mg PA) and the reaction was incubated for an additional 30 minutes at 37°C. .. For control experiments involving PA 3 with PKA or PA 1 with PKB, conditions were otherwise kept identical except for these two substitutions.

    Article Title: Murine Gammaherpesvirus 68 Open Reading Frame 45 Plays an Essential Role during the Immediate-Early Phase of Viral Replication
    Article Snippet: At 24 h p.t., cells were harvested and resuspended in dephosphorylation buffer (50 mM Tris-HCl, 10 mM MgCl2 , 100 mM NaCl, 1 mM dithiothreitol, 0.5 μM phenylmethylsulfonyl fluoride, and 1 mM benzamidine). .. After freezing and thawing twice, the mixture was sonicated and incubated with calf intestine alkaline phosphatase (New England BioLabs) (500 U/ml) at 37°C for 30 min. .. Cell lysates were subjected to Western blotting analysis with a monoclonal antibody to FLAG.

    Stripping Membranes:

    Article Title: Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines
    Article Snippet: Following IEF using a Protean IEF cell (Bio-Rad) for 10,000 volt-hours at 50 μA per strip with rapid ramping, the strips were equilibrated for 20 minutes with equilibration buffer containing 6 M urea, 2% SDS, 0.05 M Tris-HCl pH 8.8, 20% glycerol, and 2% β-mercaptoethanol, followed by SDS-PAGE (10%) and Western blot analysis. .. For treatment with calf intestinal alkaline phosphatase (CIP), cell pellets were first lysed in RIPA as described above, followed by the addition of 10 μl of 10 U/μl CIP from New England Biolabs (Ipswich, MA) in 1× New England Biolabs buffer 2.

    Expressing:

    Article Title: Long-Chain N-Acyl Amino Acid Synthases Are Linked to the Putative PEP-CTERM/Exosortase Protein-Sorting System in Gram-Negative Bacteria
    Article Snippet: PCRs were carried out under the following conditions using a Biometra TGradient thermocycler: 100-μl reaction mixtures consisted of 1.25× Thermopol buffer (NEB), 500 μM deoxynucleoside triphosphate (dNTP) mix, primers at 0.4 μM each, 100 ng of template DNA, and 0.5 μl per reaction of both Phusion Hot Start DNA Polymerase and Dynazyme II DNA Polymerase (NEB-Finnzymes); the PCR program consisted of 1 cycle at 95°C for 120 s, followed by 31 cycles of 30 s at 97°C, 30 s at 64°C, and 50 s at 72°C, followed by 120 s at 72°C. .. All PCR amplicons were subsequently digested with NdeI and PstI (except for the ExoATC3 gene amplicon, which required NcoI and SbfI) in preparation for cloning into the custom E. coli expression vector p4R-Ptac (Epoch Biolabs, Inc., Sugarland, TX), which was prepared by reciprocal enzymatic digestion and calf intestinal phosphatase (CIP) treatment (NEB). .. Insert and vector samples were ligated (Fast-Link DNA Ligation kit; Epicentre), transformed into Transformax electrocompetent E. coli EC100 (Epicentre), and selected on Luria-Bertani agar (LB-agar) containing 200 μg ml−1 ampicillin.

    Article Title: Identification of a cyclic nucleotide as a cryptic intermediate in molybdenum cofactor biosynthesis
    Article Snippet: Chemically competent E. coli DH5α and BL21(DE3) cells, and all PCR primers were from Invitrogen. pET expression plasmids were from Novagen. .. Calf-intestine alkaline phosphatase (CIAP, 20 U/μL) was from NEB.

    Article Title: The RAS-Binding Domain of Human BRAF Protein Serine/Threonine Kinase Exhibits Allosteric Conformational Changes upon Binding HRAS
    Article Snippet: Paragraph title: Cloning, Expression, Purification, and Sample Preparation ... To activate HRAS for binding to BRAF RBD, the purified bacterially expressed GDP form of HRAS was treated with calf intestinal alkaline phosphatase (New England BioLabs) in the presence of a non-hydrolyzable analog of GTP, GppNHp (Sigma) ( ).

    Article Title: Murine Gammaherpesvirus 68 Open Reading Frame 45 Plays an Essential Role during the Immediate-Early Phase of Viral Replication
    Article Snippet: 293T cells were transfected with an expression plasmid containing the full-length ORF45 fused to a FLAG tag at the N terminus. .. After freezing and thawing twice, the mixture was sonicated and incubated with calf intestine alkaline phosphatase (New England BioLabs) (500 U/ml) at 37°C for 30 min.

    Genome Wide:

    Article Title: Genome-wide assessment of sequence-intrinsic enhancer responsiveness at single-base-pair resolution
    Article Snippet: 24 h after electroporation total RNA was isolated followed by polyA+ RNA purification and DNaseI treatment, as described previously . .. 10–20 µg (focused) or 200 µg (genome-wide) of DNaseI-treated RNA was incubated with calf intestinal alkaline phosphatase (CIP; NEB cat. no. M0290L). .. Per 1 µg RNA, 0.5 µl CIP was used.

    Modification:

    Article Title: Deletion of v-chiA from a Baculovirus Reduces Horizontal Transmission in the Field
    Article Snippet: DNA from LdMNPV isolates 203-WT and 203-NL was purified from polyhedra using a modification of the method from O'Reilly et al. ( ). .. The 12.1-kbp band from isolate 203-WT and the 8.3-kbp band from 203-NL were excised from a Tris-borate-EDTA (TBE) agarose gel, purified from the agarose using the GeneClean spin kit (Bio101), and ligated into SpeI-digested pBluescriptSK+ vector after dephosphorylation using calf intestinal alkaline phosphatase (New England BioLabs).

    Western Blot:

    Article Title: Afferent Regulation of Chicken Auditory Brainstem Neurons: Rapid Changes in Phosphorylation of Elongation Factor 2
    Article Snippet: Paragraph title: Western blot ... For phosphatase treatment, HEK293 cells were incubated in a lysis buffer with Calf Intestinal Phosphatase (CIP; # M0290S; New England Biolabs, Ipswich, MA) at 1 unit CIP per 1 μg protein for 30 minutes at 37ºC before harvest.

    Article Title: Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines
    Article Snippet: Following IEF using a Protean IEF cell (Bio-Rad) for 10,000 volt-hours at 50 μA per strip with rapid ramping, the strips were equilibrated for 20 minutes with equilibration buffer containing 6 M urea, 2% SDS, 0.05 M Tris-HCl pH 8.8, 20% glycerol, and 2% β-mercaptoethanol, followed by SDS-PAGE (10%) and Western blot analysis. .. For treatment with calf intestinal alkaline phosphatase (CIP), cell pellets were first lysed in RIPA as described above, followed by the addition of 10 μl of 10 U/μl CIP from New England Biolabs (Ipswich, MA) in 1× New England Biolabs buffer 2.

    Article Title: INAUGURAL ARTICLE by a Recently Elected Academy Member:Shaggy/glycogen synthase kinase 3β and phosphorylation of Sarah/regulator of calcineurin are essential for completion of Drosophila female meiosis
    Article Snippet: Paragraph title: Western Blot. ... Calf intestinal alkaline phosphatase (CIP) treatment was performed in 1× NEBuffer 3 with the addition of 10 units CIP (NEB) for 30 min at 37 °C.

    Transformation Assay:

    Article Title: Mutants Resistant to LpxC Inhibitors by Rebalancing Cellular Homeostasis
    Article Snippet: The vector was treated with calf intestinal alkaline phosphatase (New England Biolabs). .. Correct constructs were verified using primers 10 and 11 for DNA fragment amplification and sequencing.

    High Performance Liquid Chromatography:

    Article Title: Production, Purification, and Characterization of 15N-Labeled DNA Repair Proteins as Internal Standards for Mass Spectrometric Measurements
    Article Snippet: Restriction endonucleases, DNA ligase, and calf intestinal alkaline phosphatase were obtained from New England Biolabs (MA). .. Restriction endonucleases, DNA ligase, and calf intestinal alkaline phosphatase were obtained from New England Biolabs (MA).

    Electroporation:

    Article Title: Genome-wide assessment of sequence-intrinsic enhancer responsiveness at single-base-pair resolution
    Article Snippet: 24 h after electroporation total RNA was isolated followed by polyA+ RNA purification and DNaseI treatment, as described previously . .. 10–20 µg (focused) or 200 µg (genome-wide) of DNaseI-treated RNA was incubated with calf intestinal alkaline phosphatase (CIP; NEB cat. no. M0290L).

    Transfection:

    Article Title: Murine Gammaherpesvirus 68 Open Reading Frame 45 Plays an Essential Role during the Immediate-Early Phase of Viral Replication
    Article Snippet: 293T cells were transfected with an expression plasmid containing the full-length ORF45 fused to a FLAG tag at the N terminus. .. After freezing and thawing twice, the mixture was sonicated and incubated with calf intestine alkaline phosphatase (New England BioLabs) (500 U/ml) at 37°C for 30 min.

    Immunoprecipitation:

    Article Title: Transcriptome-wide microRNA and target dynamics in the fat body during the gonadotrophic cycle of Aedes aegypti
    Article Snippet: GenScript custom AGO1 antibody (lot no. A313070458) was bound to Protein A Dynabeads (Invitrogen) and immunoprecipitation was performed overnight at 4 °C. .. Beads were further digested with 1:1,000 RNase A dilution, dephosphorylated with calf intestinal alkaline phosphatase (CIP) (NEB) and ligated to 32 P-labeled 3′ RNA linker (RL3-OH) using T4 RNA ligase (Thermo Fisher Scientific).

    Protease Inhibitor:

    Article Title: Afferent Regulation of Chicken Auditory Brainstem Neurons: Rapid Changes in Phosphorylation of Elongation Factor 2
    Article Snippet: For phosphatase treatment, HEK293 cells were incubated in a lysis buffer with Calf Intestinal Phosphatase (CIP; # M0290S; New England Biolabs, Ipswich, MA) at 1 unit CIP per 1 μg protein for 30 minutes at 37ºC before harvest. .. For phosphatase treatment, HEK293 cells were incubated in a lysis buffer with Calf Intestinal Phosphatase (CIP; # M0290S; New England Biolabs, Ipswich, MA) at 1 unit CIP per 1 μg protein for 30 minutes at 37ºC before harvest.

    DNA Sequencing:

    Article Title: Cloning, Expression, and Site-Directed Mutagenesis of the Propene Monooxygenase Genes from Mycobacterium sp. Strain M156
    Article Snippet: Restriction enzymes and DNA-modifying enzymes (calf intestinal alkaline phosphatase and DNA ligase) were purchased from New England Biolabs and Roche Diagnostics Ltd., respectively. .. Restriction enzymes and DNA-modifying enzymes (calf intestinal alkaline phosphatase and DNA ligase) were purchased from New England Biolabs and Roche Diagnostics Ltd., respectively.

    Sequencing:

    Article Title: Deletion of v-chiA from a Baculovirus Reduces Horizontal Transmission in the Field
    Article Snippet: Paragraph title: Cloning, sequence analysis, and generation of recombinant viruses. ... The 12.1-kbp band from isolate 203-WT and the 8.3-kbp band from 203-NL were excised from a Tris-borate-EDTA (TBE) agarose gel, purified from the agarose using the GeneClean spin kit (Bio101), and ligated into SpeI-digested pBluescriptSK+ vector after dephosphorylation using calf intestinal alkaline phosphatase (New England BioLabs).

    Article Title: Cloning, Expression, and Site-Directed Mutagenesis of the Propene Monooxygenase Genes from Mycobacterium sp. Strain M156
    Article Snippet: Restriction enzymes and DNA-modifying enzymes (calf intestinal alkaline phosphatase and DNA ligase) were purchased from New England Biolabs and Roche Diagnostics Ltd., respectively. .. Restriction enzymes and DNA-modifying enzymes (calf intestinal alkaline phosphatase and DNA ligase) were purchased from New England Biolabs and Roche Diagnostics Ltd., respectively.

    Article Title: Mutants Resistant to LpxC Inhibitors by Rebalancing Cellular Homeostasis
    Article Snippet: The vector was treated with calf intestinal alkaline phosphatase (New England Biolabs). .. After PCR purification, the vector and DNA fragment were ligated using T4 DNA ligase (Invitrogen), transformed into XL1-Blue Competent cells (Stratagene, Santa Clara, CA), and grown on LB agar containing 25 μg/ml chloramphenicol (Sigma).

    Article Title: Requirement of the galU Gene for Polysaccharide Production by and Pathogenicity and Growth In Planta of Xanthomonas citri subsp. citri
    Article Snippet: The entire galU gene with 624 bp of upstream sequence and 388 bp of downstream sequence was amplified from genomic DNA of X. citri subsp. citri wild-type strain 306 by a PCR performed with primers CGU-F and CGU-R, which contained a BamHI restriction site (Table ). .. A BamHI fragment containing the galU gene was isolated from pCGU1.1 and cloned into similarly digested pUFR053 that was treated with calf intestinal alkaline phosphatase (New England Biolabs, Ipswich, MA) , resulting in pCGU2.1 (Table ).

    Sonication:

    Article Title: Murine Gammaherpesvirus 68 Open Reading Frame 45 Plays an Essential Role during the Immediate-Early Phase of Viral Replication
    Article Snippet: At 24 h p.t., cells were harvested and resuspended in dephosphorylation buffer (50 mM Tris-HCl, 10 mM MgCl2 , 100 mM NaCl, 1 mM dithiothreitol, 0.5 μM phenylmethylsulfonyl fluoride, and 1 mM benzamidine). .. After freezing and thawing twice, the mixture was sonicated and incubated with calf intestine alkaline phosphatase (New England BioLabs) (500 U/ml) at 37°C for 30 min. .. Cell lysates were subjected to Western blotting analysis with a monoclonal antibody to FLAG.

    Binding Assay:

    Article Title: The RAS-Binding Domain of Human BRAF Protein Serine/Threonine Kinase Exhibits Allosteric Conformational Changes upon Binding HRAS
    Article Snippet: Tagless and unlabeled HRAS used for binding studies with BRAF RBD was expressed in E. coli Tuner(DE3) cells (EMD Millipore) grown in LB medium and cleaved using N-terminal hexaHis-tagged TEV protease ( ). .. To activate HRAS for binding to BRAF RBD, the purified bacterially expressed GDP form of HRAS was treated with calf intestinal alkaline phosphatase (New England BioLabs) in the presence of a non-hydrolyzable analog of GTP, GppNHp (Sigma) ( ). .. Samples of [ U -13 C,15 N]- and [ U -5%-13 C,100%-15 N]-BRAF RBD for NMR structure determination were concentrated by centrifugation to 0.7–0.9 mM in 20 mM ammonium acetate, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2 , 50 µM 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS), and 10% 2 H2 O (v/v) at pH 4.5.

    Article Title: Polyphosphate/platelet factor 4 complexes can mediate heparin-independent platelet activation in heparin-induced thrombocytopenia
    Article Snippet: The following were purchased from commercial sources: thrombin receptor agonist peptide (TRAP), prostaglandin E1 (PGE1), and chondroitinase ABC from Proteus vulgaris (Sigma); calf intestinal alkaline phosphatase (CIP; New England Biolabs, Ipswich, MA); unfractionated heparin (UFH; Becton-Dickenson, Franklin Lakes, NJ); Hanks balanced salt solution (HBSS) and phosphate-buffered saline (Invitrogen/Life Technologies and GIBCO-Life Sciences, Grand Island, MI); gelatin veronal buffer, normal human serum (NHS) and human complement factors (Complement Technology, Inc., Tyler, TX); 96-well microplates (Corning, Amsterdam, Netherlands); 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Roche Diagnostics, Indianapolis, IN); 0.22-µM filters (Millipore); plastic cuvettes (Fisher); horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc and HRP-conjugated goat anti-mouse IgG Fc antibodies (Jackson ImmunoResearch; West Grove, PA); HRP-conjugated goat anti-human C3 antibody (MP Biomedicals; Santa Ana, CA) that recognizes native, hydrolyzed C3b, iC3b, C3c, and C3d; and chicken erythrocytes (Colorado Serum Company, Denver, CO). .. The following were purchased from commercial sources: thrombin receptor agonist peptide (TRAP), prostaglandin E1 (PGE1), and chondroitinase ABC from Proteus vulgaris (Sigma); calf intestinal alkaline phosphatase (CIP; New England Biolabs, Ipswich, MA); unfractionated heparin (UFH; Becton-Dickenson, Franklin Lakes, NJ); Hanks balanced salt solution (HBSS) and phosphate-buffered saline (Invitrogen/Life Technologies and GIBCO-Life Sciences, Grand Island, MI); gelatin veronal buffer, normal human serum (NHS) and human complement factors (Complement Technology, Inc., Tyler, TX); 96-well microplates (Corning, Amsterdam, Netherlands); 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Roche Diagnostics, Indianapolis, IN); 0.22-µM filters (Millipore); plastic cuvettes (Fisher); horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc and HRP-conjugated goat anti-mouse IgG Fc antibodies (Jackson ImmunoResearch; West Grove, PA); HRP-conjugated goat anti-human C3 antibody (MP Biomedicals; Santa Ana, CA) that recognizes native, hydrolyzed C3b, iC3b, C3c, and C3d; and chicken erythrocytes (Colorado Serum Company, Denver, CO).

    De-Phosphorylation Assay:

    Article Title: Deletion of v-chiA from a Baculovirus Reduces Horizontal Transmission in the Field
    Article Snippet: This area of the genome contains the chitinase gene (bp 64801 to 66477) based on the LdMNPV 5-6 sequence ( ). .. The 12.1-kbp band from isolate 203-WT and the 8.3-kbp band from 203-NL were excised from a Tris-borate-EDTA (TBE) agarose gel, purified from the agarose using the GeneClean spin kit (Bio101), and ligated into SpeI-digested pBluescriptSK+ vector after dephosphorylation using calf intestinal alkaline phosphatase (New England BioLabs). .. The ligation mix was used to transform competent DH5α cells using standard techniques.

    Article Title: Amyloid-β oligomer Aβ*56 induces specific alterations of tau phosphorylation and neuronal signaling
    Article Snippet: Paragraph title: Tau dephosphorylation ... Tau (50 μg IC lysate/reaction) was dephosphorylated by treatment with calf-intestinal alkaline phosphatase (CIP, New England BioLabs, Inc.) at 20 units/mL for 3 hours at 37°C.

    Article Title: Murine Gammaherpesvirus 68 Open Reading Frame 45 Plays an Essential Role during the Immediate-Early Phase of Viral Replication
    Article Snippet: Paragraph title: Dephosphorylation assay. ... After freezing and thawing twice, the mixture was sonicated and incubated with calf intestine alkaline phosphatase (New England BioLabs) (500 U/ml) at 37°C for 30 min.

    Molecular Weight:

    Article Title: Afferent Regulation of Chicken Auditory Brainstem Neurons: Rapid Changes in Phosphorylation of Elongation Factor 2
    Article Snippet: For phosphatase treatment, HEK293 cells were incubated in a lysis buffer with Calf Intestinal Phosphatase (CIP; # M0290S; New England Biolabs, Ipswich, MA) at 1 unit CIP per 1 μg protein for 30 minutes at 37ºC before harvest. .. For phosphatase treatment, HEK293 cells were incubated in a lysis buffer with Calf Intestinal Phosphatase (CIP; # M0290S; New England Biolabs, Ipswich, MA) at 1 unit CIP per 1 μg protein for 30 minutes at 37ºC before harvest.

    Liquid Chromatography with Mass Spectroscopy:

    Article Title: Switching of Self-Assembly in a Peptide Nanostructure with a Specific Enzyme
    Article Snippet: For the LC-MS, cryogenic-TEM, SAXS, and CD experiments the PA was dissolved at 1 mM in a reaction buffer of 50 mM Tris-HCl and 10 mM MgCl2 (pH=7.5). .. Following this time, calf intestinal alkaline phosphatase (CIP, New England Biolabs) was added (60 U/mg PA) and the reaction was incubated for an additional 30 minutes at 37°C.

    Isolation:

    Article Title: Genome-wide assessment of sequence-intrinsic enhancer responsiveness at single-base-pair resolution
    Article Snippet: 24 h after electroporation total RNA was isolated followed by polyA+ RNA purification and DNaseI treatment, as described previously . .. 10–20 µg (focused) or 200 µg (genome-wide) of DNaseI-treated RNA was incubated with calf intestinal alkaline phosphatase (CIP; NEB cat. no. M0290L).

    Article Title: Requirement of the galU Gene for Polysaccharide Production by and Pathogenicity and Growth In Planta of Xanthomonas citri subsp. citri
    Article Snippet: The resulting 1.9-kb fragment was ligated to PCR 2.1-TOPO using the manufacturer's protocol (Invitrogen, Carlsbad, CA), resulting in pCGU1.1. .. A BamHI fragment containing the galU gene was isolated from pCGU1.1 and cloned into similarly digested pUFR053 that was treated with calf intestinal alkaline phosphatase (New England Biolabs, Ipswich, MA) , resulting in pCGU2.1 (Table ). .. Plasmid pCGU2.1 was transferred into galU mutants D12 and F6 ( galU ::Tn 5 ) by triparental mating with an E. coli helper strain containing pRK2013 ( ).

    Subcloning:

    Article Title: Cloning, Expression, and Site-Directed Mutagenesis of the Propene Monooxygenase Genes from Mycobacterium sp. Strain M156
    Article Snippet: Restriction enzymes and DNA-modifying enzymes (calf intestinal alkaline phosphatase and DNA ligase) were purchased from New England Biolabs and Roche Diagnostics Ltd., respectively. .. High-purity plasmid DNAs were purified from E. coli clones by the use of QIAprep Spin miniprep kits (QIAGEN), while DNAs were recovered from agarose gels by use of a DNA Wizard clean-up kit from Promega or a QIAQuick gel extraction kit from QIAGEN.

    Flow Cytometry:

    Article Title: Polyphosphate/platelet factor 4 complexes can mediate heparin-independent platelet activation in heparin-induced thrombocytopenia
    Article Snippet: Here we characterize the biophysical, antigenic, and platelet-activating properties of PF4/polyP complexes and ask whether activated platelets can generate endogenous polyP-containing antigenic complexes capable of exacerbating and perhaps perpetuating HIT in the absence of exogenous PF4 and heparin. .. The following were purchased from commercial sources: thrombin receptor agonist peptide (TRAP), prostaglandin E1 (PGE1), and chondroitinase ABC from Proteus vulgaris (Sigma); calf intestinal alkaline phosphatase (CIP; New England Biolabs, Ipswich, MA); unfractionated heparin (UFH; Becton-Dickenson, Franklin Lakes, NJ); Hanks balanced salt solution (HBSS) and phosphate-buffered saline (Invitrogen/Life Technologies and GIBCO-Life Sciences, Grand Island, MI); gelatin veronal buffer, normal human serum (NHS) and human complement factors (Complement Technology, Inc., Tyler, TX); 96-well microplates (Corning, Amsterdam, Netherlands); 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Roche Diagnostics, Indianapolis, IN); 0.22-µM filters (Millipore); plastic cuvettes (Fisher); horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc and HRP-conjugated goat anti-mouse IgG Fc antibodies (Jackson ImmunoResearch; West Grove, PA); HRP-conjugated goat anti-human C3 antibody (MP Biomedicals; Santa Ana, CA) that recognizes native, hydrolyzed C3b, iC3b, C3c, and C3d; and chicken erythrocytes (Colorado Serum Company, Denver, CO). .. CIP was buffer exchanged using Micro Bio-Spin Columns with Bio-Gel P-6 (Bio-Rad ) .

    Labeling:

    Article Title: Afferent Regulation of Chicken Auditory Brainstem Neurons: Rapid Changes in Phosphorylation of Elongation Factor 2
    Article Snippet: For phosphatase treatment, HEK293 cells were incubated in a lysis buffer with Calf Intestinal Phosphatase (CIP; # M0290S; New England Biolabs, Ipswich, MA) at 1 unit CIP per 1 μg protein for 30 minutes at 37ºC before harvest. .. For phosphatase treatment, HEK293 cells were incubated in a lysis buffer with Calf Intestinal Phosphatase (CIP; # M0290S; New England Biolabs, Ipswich, MA) at 1 unit CIP per 1 μg protein for 30 minutes at 37ºC before harvest.

    Article Title: Deformability in the Cleavage Site of Primary MicroRNA is Not Sensed by the Double-Stranded RNA Binding Domains in the Microprocessor Component DGCR8
    Article Snippet: Preparation of the template DNA, transcription by T7 RNA polymerase, and purification of the transcribed RNA were all performed as previously described. .. All RNAs were 5′-end labeled with 32 P. In order to remove the 5′-triphosphate, the RNA was first treated with calf intestinal alkaline phosphatase (New England Biolabs), phenol/chloroform extracted, and ethanol precipitated. .. The RNA was then 5′-end labeled with T4 polynucleotide kinase (New England Biolabs).

    Purification:

    Article Title: Genome-wide assessment of sequence-intrinsic enhancer responsiveness at single-base-pair resolution
    Article Snippet: 24 h after electroporation total RNA was isolated followed by polyA+ RNA purification and DNaseI treatment, as described previously . .. 10–20 µg (focused) or 200 µg (genome-wide) of DNaseI-treated RNA was incubated with calf intestinal alkaline phosphatase (CIP; NEB cat. no. M0290L).

    Article Title: Deletion of v-chiA from a Baculovirus Reduces Horizontal Transmission in the Field
    Article Snippet: This area of the genome contains the chitinase gene (bp 64801 to 66477) based on the LdMNPV 5-6 sequence ( ). .. The 12.1-kbp band from isolate 203-WT and the 8.3-kbp band from 203-NL were excised from a Tris-borate-EDTA (TBE) agarose gel, purified from the agarose using the GeneClean spin kit (Bio101), and ligated into SpeI-digested pBluescriptSK+ vector after dephosphorylation using calf intestinal alkaline phosphatase (New England BioLabs). .. The ligation mix was used to transform competent DH5α cells using standard techniques.

    Article Title: Mutants Resistant to LpxC Inhibitors by Rebalancing Cellular Homeostasis
    Article Snippet: The PCR fragment was purified using QIAQuick gel extraction kit (Qiagen, Valencia, CA). .. The vector was treated with calf intestinal alkaline phosphatase (New England Biolabs).

    Article Title: Ubp-M serine 552 phosphorylation by cyclin-dependent kinase 1 regulates cell cycle progression
    Article Snippet: Ubp-M purified from sf9 cells appears as a doublet on SDS-PAGE gels, similar to Ubp-M purified from HeLa cells ( , lane 2, and data not shown). .. For this purpose, we treated Ubp-M with Calf Intestinal Alkaline Phosphatase (CIP, NEB).

    Article Title: The RAS-Binding Domain of Human BRAF Protein Serine/Threonine Kinase Exhibits Allosteric Conformational Changes upon Binding HRAS
    Article Snippet: Tagless and unlabeled HRAS used for binding studies with BRAF RBD was expressed in E. coli Tuner(DE3) cells (EMD Millipore) grown in LB medium and cleaved using N-terminal hexaHis-tagged TEV protease ( ). .. To activate HRAS for binding to BRAF RBD, the purified bacterially expressed GDP form of HRAS was treated with calf intestinal alkaline phosphatase (New England BioLabs) in the presence of a non-hydrolyzable analog of GTP, GppNHp (Sigma) ( ). .. Samples of [ U -13 C,15 N]- and [ U -5%-13 C,100%-15 N]-BRAF RBD for NMR structure determination were concentrated by centrifugation to 0.7–0.9 mM in 20 mM ammonium acetate, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2 , 50 µM 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS), and 10% 2 H2 O (v/v) at pH 4.5.

    Article Title: Polyphosphate/platelet factor 4 complexes can mediate heparin-independent platelet activation in heparin-induced thrombocytopenia
    Article Snippet: The following were purchased from commercial sources: thrombin receptor agonist peptide (TRAP), prostaglandin E1 (PGE1), and chondroitinase ABC from Proteus vulgaris (Sigma); calf intestinal alkaline phosphatase (CIP; New England Biolabs, Ipswich, MA); unfractionated heparin (UFH; Becton-Dickenson, Franklin Lakes, NJ); Hanks balanced salt solution (HBSS) and phosphate-buffered saline (Invitrogen/Life Technologies and GIBCO-Life Sciences, Grand Island, MI); gelatin veronal buffer, normal human serum (NHS) and human complement factors (Complement Technology, Inc., Tyler, TX); 96-well microplates (Corning, Amsterdam, Netherlands); 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Roche Diagnostics, Indianapolis, IN); 0.22-µM filters (Millipore); plastic cuvettes (Fisher); horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc and HRP-conjugated goat anti-mouse IgG Fc antibodies (Jackson ImmunoResearch; West Grove, PA); HRP-conjugated goat anti-human C3 antibody (MP Biomedicals; Santa Ana, CA) that recognizes native, hydrolyzed C3b, iC3b, C3c, and C3d; and chicken erythrocytes (Colorado Serum Company, Denver, CO). .. The following were purchased from commercial sources: thrombin receptor agonist peptide (TRAP), prostaglandin E1 (PGE1), and chondroitinase ABC from Proteus vulgaris (Sigma); calf intestinal alkaline phosphatase (CIP; New England Biolabs, Ipswich, MA); unfractionated heparin (UFH; Becton-Dickenson, Franklin Lakes, NJ); Hanks balanced salt solution (HBSS) and phosphate-buffered saline (Invitrogen/Life Technologies and GIBCO-Life Sciences, Grand Island, MI); gelatin veronal buffer, normal human serum (NHS) and human complement factors (Complement Technology, Inc., Tyler, TX); 96-well microplates (Corning, Amsterdam, Netherlands); 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Roche Diagnostics, Indianapolis, IN); 0.22-µM filters (Millipore); plastic cuvettes (Fisher); horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc and HRP-conjugated goat anti-mouse IgG Fc antibodies (Jackson ImmunoResearch; West Grove, PA); HRP-conjugated goat anti-human C3 antibody (MP Biomedicals; Santa Ana, CA) that recognizes native, hydrolyzed C3b, iC3b, C3c, and C3d; and chicken erythrocytes (Colorado Serum Company, Denver, CO).

    Polymerase Chain Reaction:

    Article Title: Long-Chain N-Acyl Amino Acid Synthases Are Linked to the Putative PEP-CTERM/Exosortase Protein-Sorting System in Gram-Negative Bacteria
    Article Snippet: PCRs were carried out under the following conditions using a Biometra TGradient thermocycler: 100-μl reaction mixtures consisted of 1.25× Thermopol buffer (NEB), 500 μM deoxynucleoside triphosphate (dNTP) mix, primers at 0.4 μM each, 100 ng of template DNA, and 0.5 μl per reaction of both Phusion Hot Start DNA Polymerase and Dynazyme II DNA Polymerase (NEB-Finnzymes); the PCR program consisted of 1 cycle at 95°C for 120 s, followed by 31 cycles of 30 s at 97°C, 30 s at 64°C, and 50 s at 72°C, followed by 120 s at 72°C. .. All PCR amplicons were subsequently digested with NdeI and PstI (except for the ExoATC3 gene amplicon, which required NcoI and SbfI) in preparation for cloning into the custom E. coli expression vector p4R-Ptac (Epoch Biolabs, Inc., Sugarland, TX), which was prepared by reciprocal enzymatic digestion and calf intestinal phosphatase (CIP) treatment (NEB). .. Insert and vector samples were ligated (Fast-Link DNA Ligation kit; Epicentre), transformed into Transformax electrocompetent E. coli EC100 (Epicentre), and selected on Luria-Bertani agar (LB-agar) containing 200 μg ml−1 ampicillin.

    Article Title: Mutants Resistant to LpxC Inhibitors by Rebalancing Cellular Homeostasis
    Article Snippet: Both the vector and PCR fragment were digested using restriction enzymes NdeI and HindIII (New England Biolabs, Ipswich, MA). .. The vector was treated with calf intestinal alkaline phosphatase (New England Biolabs).

    Article Title: Identification of a cyclic nucleotide as a cryptic intermediate in molybdenum cofactor biosynthesis
    Article Snippet: Chemically competent E. coli DH5α and BL21(DE3) cells, and all PCR primers were from Invitrogen. pET expression plasmids were from Novagen. .. Calf-intestine alkaline phosphatase (CIAP, 20 U/μL) was from NEB.

    Positron Emission Tomography:

    Article Title: Identification of a cyclic nucleotide as a cryptic intermediate in molybdenum cofactor biosynthesis
    Article Snippet: Chemically competent E. coli DH5α and BL21(DE3) cells, and all PCR primers were from Invitrogen. pET expression plasmids were from Novagen. .. Calf-intestine alkaline phosphatase (CIAP, 20 U/μL) was from NEB.

    Article Title: The RAS-Binding Domain of Human BRAF Protein Serine/Threonine Kinase Exhibits Allosteric Conformational Changes upon Binding HRAS
    Article Snippet: To activate HRAS for binding to BRAF RBD, the purified bacterially expressed GDP form of HRAS was treated with calf intestinal alkaline phosphatase (New England BioLabs) in the presence of a non-hydrolyzable analog of GTP, GppNHp (Sigma) ( ). .. Samples of 5-F-Trp BRAF RBD for 19 F NMR spectroscopy were concentrated to 0.2–0.5 mM in pH 7.5 buffer (see above).

    Spectroscopy:

    Article Title: The RAS-Binding Domain of Human BRAF Protein Serine/Threonine Kinase Exhibits Allosteric Conformational Changes upon Binding HRAS
    Article Snippet: In brief, isotopically enriched BRAF RBD for NMR spectroscopy and X-ray crystallography (BRAF[149–232] and BRAF[153–237], respectively), each containing an 11-residue N-terminal affinity tag (MGHHHHHHSHM), were expressed in Escherichia coli BL21(DE3)-Gold cells (Agilent) grown in MJ9 minimal medium ( ) containing U -(15 NH4 )2 SO4 and [ U -13 C]-glucose as the sole nitrogen and carbon sources for NMR, or supplemented with selenomethionine for crystallography ( ). .. To activate HRAS for binding to BRAF RBD, the purified bacterially expressed GDP form of HRAS was treated with calf intestinal alkaline phosphatase (New England BioLabs) in the presence of a non-hydrolyzable analog of GTP, GppNHp (Sigma) ( ).

    Lysis:

    Article Title: Afferent Regulation of Chicken Auditory Brainstem Neurons: Rapid Changes in Phosphorylation of Elongation Factor 2
    Article Snippet: A western blot immunoassay was conducted to confirm the specificity of anti-eEF2 and anti-p-eEF2 used in the present study. .. For phosphatase treatment, HEK293 cells were incubated in a lysis buffer with Calf Intestinal Phosphatase (CIP; # M0290S; New England Biolabs, Ipswich, MA) at 1 unit CIP per 1 μg protein for 30 minutes at 37ºC before harvest. .. Brain protein samples were harvested from the NM and the surrounding region in the dorsocaudal brainstem of chicks.

    Article Title: Transcriptome-wide microRNA and target dynamics in the fat body during the gonadotrophic cycle of Aedes aegypti
    Article Snippet: Following tissue lysis, lysates were treated with RQ1 DNase (Promega) and 1:1,000 RNase A (Ambion) dilution. .. Beads were further digested with 1:1,000 RNase A dilution, dephosphorylated with calf intestinal alkaline phosphatase (CIP) (NEB) and ligated to 32 P-labeled 3′ RNA linker (RL3-OH) using T4 RNA ligase (Thermo Fisher Scientific).

    Concentration Assay:

    Article Title: Deformability in the Cleavage Site of Primary MicroRNA is Not Sensed by the Double-Stranded RNA Binding Domains in the Microprocessor Component DGCR8
    Article Snippet: All RNAs were 5′-end labeled with 32 P. In order to remove the 5′-triphosphate, the RNA was first treated with calf intestinal alkaline phosphatase (New England Biolabs), phenol/chloroform extracted, and ethanol precipitated. .. The RNA was then 5′-end labeled with T4 polynucleotide kinase (New England Biolabs).

    Article Title: Identification of a cyclic nucleotide as a cryptic intermediate in molybdenum cofactor biosynthesis
    Article Snippet: Calf-intestine alkaline phosphatase (CIAP, 20 U/μL) was from NEB. .. Non-linear least square fitting of kinetic data was carried out using KaleidaGraph software (Synergy Software, Reading, PA).

    Article Title: The RAS-Binding Domain of Human BRAF Protein Serine/Threonine Kinase Exhibits Allosteric Conformational Changes upon Binding HRAS
    Article Snippet: To activate HRAS for binding to BRAF RBD, the purified bacterially expressed GDP form of HRAS was treated with calf intestinal alkaline phosphatase (New England BioLabs) in the presence of a non-hydrolyzable analog of GTP, GppNHp (Sigma) ( ). .. Samples of [ U -13 C,15 N]- and [ U -5%-13 C,100%-15 N]-BRAF RBD for NMR structure determination were concentrated by centrifugation to 0.7–0.9 mM in 20 mM ammonium acetate, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2 , 50 µM 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS), and 10% 2 H2 O (v/v) at pH 4.5.

    Article Title: INAUGURAL ARTICLE by a Recently Elected Academy Member:Shaggy/glycogen synthase kinase 3β and phosphorylation of Sarah/regulator of calcineurin are essential for completion of Drosophila female meiosis
    Article Snippet: Calf intestinal alkaline phosphatase (CIP) treatment was performed in 1× NEBuffer 3 with the addition of 10 units CIP (NEB) for 30 min at 37 °C. .. Calf intestinal alkaline phosphatase (CIP) treatment was performed in 1× NEBuffer 3 with the addition of 10 units CIP (NEB) for 30 min at 37 °C.

    Agarose Gel Electrophoresis:

    Article Title: Deletion of v-chiA from a Baculovirus Reduces Horizontal Transmission in the Field
    Article Snippet: This area of the genome contains the chitinase gene (bp 64801 to 66477) based on the LdMNPV 5-6 sequence ( ). .. The 12.1-kbp band from isolate 203-WT and the 8.3-kbp band from 203-NL were excised from a Tris-borate-EDTA (TBE) agarose gel, purified from the agarose using the GeneClean spin kit (Bio101), and ligated into SpeI-digested pBluescriptSK+ vector after dephosphorylation using calf intestinal alkaline phosphatase (New England BioLabs). .. The ligation mix was used to transform competent DH5α cells using standard techniques.

    SDS Page:

    Article Title: Ubp-M serine 552 phosphorylation by cyclin-dependent kinase 1 regulates cell cycle progression
    Article Snippet: Ubp-M purified from sf9 cells appears as a doublet on SDS-PAGE gels, similar to Ubp-M purified from HeLa cells ( , lane 2, and data not shown). .. For this purpose, we treated Ubp-M with Calf Intestinal Alkaline Phosphatase (CIP, NEB).

    Article Title: Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines
    Article Snippet: Following IEF using a Protean IEF cell (Bio-Rad) for 10,000 volt-hours at 50 μA per strip with rapid ramping, the strips were equilibrated for 20 minutes with equilibration buffer containing 6 M urea, 2% SDS, 0.05 M Tris-HCl pH 8.8, 20% glycerol, and 2% β-mercaptoethanol, followed by SDS-PAGE (10%) and Western blot analysis. .. For treatment with calf intestinal alkaline phosphatase (CIP), cell pellets were first lysed in RIPA as described above, followed by the addition of 10 μl of 10 U/μl CIP from New England Biolabs (Ipswich, MA) in 1× New England Biolabs buffer 2.

    Plasmid Preparation:

    Article Title: Long-Chain N-Acyl Amino Acid Synthases Are Linked to the Putative PEP-CTERM/Exosortase Protein-Sorting System in Gram-Negative Bacteria
    Article Snippet: PCRs were carried out under the following conditions using a Biometra TGradient thermocycler: 100-μl reaction mixtures consisted of 1.25× Thermopol buffer (NEB), 500 μM deoxynucleoside triphosphate (dNTP) mix, primers at 0.4 μM each, 100 ng of template DNA, and 0.5 μl per reaction of both Phusion Hot Start DNA Polymerase and Dynazyme II DNA Polymerase (NEB-Finnzymes); the PCR program consisted of 1 cycle at 95°C for 120 s, followed by 31 cycles of 30 s at 97°C, 30 s at 64°C, and 50 s at 72°C, followed by 120 s at 72°C. .. All PCR amplicons were subsequently digested with NdeI and PstI (except for the ExoATC3 gene amplicon, which required NcoI and SbfI) in preparation for cloning into the custom E. coli expression vector p4R-Ptac (Epoch Biolabs, Inc., Sugarland, TX), which was prepared by reciprocal enzymatic digestion and calf intestinal phosphatase (CIP) treatment (NEB). .. Insert and vector samples were ligated (Fast-Link DNA Ligation kit; Epicentre), transformed into Transformax electrocompetent E. coli EC100 (Epicentre), and selected on Luria-Bertani agar (LB-agar) containing 200 μg ml−1 ampicillin.

    Article Title: Deletion of v-chiA from a Baculovirus Reduces Horizontal Transmission in the Field
    Article Snippet: This area of the genome contains the chitinase gene (bp 64801 to 66477) based on the LdMNPV 5-6 sequence ( ). .. The 12.1-kbp band from isolate 203-WT and the 8.3-kbp band from 203-NL were excised from a Tris-borate-EDTA (TBE) agarose gel, purified from the agarose using the GeneClean spin kit (Bio101), and ligated into SpeI-digested pBluescriptSK+ vector after dephosphorylation using calf intestinal alkaline phosphatase (New England BioLabs). .. The ligation mix was used to transform competent DH5α cells using standard techniques.

    Article Title: Mutants Resistant to LpxC Inhibitors by Rebalancing Cellular Homeostasis
    Article Snippet: Both the vector and PCR fragment were digested using restriction enzymes NdeI and HindIII (New England Biolabs, Ipswich, MA). .. The vector was treated with calf intestinal alkaline phosphatase (New England Biolabs). .. After PCR purification, the vector and DNA fragment were ligated using T4 DNA ligase (Invitrogen), transformed into XL1-Blue Competent cells (Stratagene, Santa Clara, CA), and grown on LB agar containing 25 μg/ml chloramphenicol (Sigma).

    Article Title: Requirement of the galU Gene for Polysaccharide Production by and Pathogenicity and Growth In Planta of Xanthomonas citri subsp. citri
    Article Snippet: A BamHI fragment containing the galU gene was isolated from pCGU1.1 and cloned into similarly digested pUFR053 that was treated with calf intestinal alkaline phosphatase (New England Biolabs, Ipswich, MA) , resulting in pCGU2.1 (Table ). .. The presence of pCGU2.1 was verified using PCR.

    Article Title: Murine Gammaherpesvirus 68 Open Reading Frame 45 Plays an Essential Role during the Immediate-Early Phase of Viral Replication
    Article Snippet: 293T cells were transfected with an expression plasmid containing the full-length ORF45 fused to a FLAG tag at the N terminus. .. After freezing and thawing twice, the mixture was sonicated and incubated with calf intestine alkaline phosphatase (New England BioLabs) (500 U/ml) at 37°C for 30 min.

    Software:

    Article Title: Identification of a cyclic nucleotide as a cryptic intermediate in molybdenum cofactor biosynthesis
    Article Snippet: Calf-intestine alkaline phosphatase (CIAP, 20 U/μL) was from NEB. .. UV-vis absorption spectra were determined using a U-3900 UV-VIS ratio recording double-beam spectrometer (HITACHI) or Nanodrop 1000 (Thermo Scientific).

    Irradiation:

    Article Title: Transcriptome-wide microRNA and target dynamics in the fat body during the gonadotrophic cycle of Aedes aegypti
    Article Snippet: Briefly, 60 FBs were collected at 72 h PE and 24 h PBM each, finely sliced, and irradiated to 900 mJ/cm2 . .. Beads were further digested with 1:1,000 RNase A dilution, dephosphorylated with calf intestinal alkaline phosphatase (CIP) (NEB) and ligated to 32 P-labeled 3′ RNA linker (RL3-OH) using T4 RNA ligase (Thermo Fisher Scientific).

    Recombinant:

    Article Title: Deletion of v-chiA from a Baculovirus Reduces Horizontal Transmission in the Field
    Article Snippet: Paragraph title: Cloning, sequence analysis, and generation of recombinant viruses. ... The 12.1-kbp band from isolate 203-WT and the 8.3-kbp band from 203-NL were excised from a Tris-borate-EDTA (TBE) agarose gel, purified from the agarose using the GeneClean spin kit (Bio101), and ligated into SpeI-digested pBluescriptSK+ vector after dephosphorylation using calf intestinal alkaline phosphatase (New England BioLabs).

    Sample Prep:

    Article Title: The RAS-Binding Domain of Human BRAF Protein Serine/Threonine Kinase Exhibits Allosteric Conformational Changes upon Binding HRAS
    Article Snippet: Paragraph title: Cloning, Expression, Purification, and Sample Preparation ... To activate HRAS for binding to BRAF RBD, the purified bacterially expressed GDP form of HRAS was treated with calf intestinal alkaline phosphatase (New England BioLabs) in the presence of a non-hydrolyzable analog of GTP, GppNHp (Sigma) ( ).

    Nuclear Magnetic Resonance:

    Article Title: The RAS-Binding Domain of Human BRAF Protein Serine/Threonine Kinase Exhibits Allosteric Conformational Changes upon Binding HRAS
    Article Snippet: In brief, isotopically enriched BRAF RBD for NMR spectroscopy and X-ray crystallography (BRAF[149–232] and BRAF[153–237], respectively), each containing an 11-residue N-terminal affinity tag (MGHHHHHHSHM), were expressed in Escherichia coli BL21(DE3)-Gold cells (Agilent) grown in MJ9 minimal medium ( ) containing U -(15 NH4 )2 SO4 and [ U -13 C]-glucose as the sole nitrogen and carbon sources for NMR, or supplemented with selenomethionine for crystallography ( ). .. To activate HRAS for binding to BRAF RBD, the purified bacterially expressed GDP form of HRAS was treated with calf intestinal alkaline phosphatase (New England BioLabs) in the presence of a non-hydrolyzable analog of GTP, GppNHp (Sigma) ( ).

    FLAG-tag:

    Article Title: Murine Gammaherpesvirus 68 Open Reading Frame 45 Plays an Essential Role during the Immediate-Early Phase of Viral Replication
    Article Snippet: 293T cells were transfected with an expression plasmid containing the full-length ORF45 fused to a FLAG tag at the N terminus. .. After freezing and thawing twice, the mixture was sonicated and incubated with calf intestine alkaline phosphatase (New England BioLabs) (500 U/ml) at 37°C for 30 min.

    Two-Dimensional Gel Electrophoresis:

    Article Title: Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines
    Article Snippet: Paragraph title: Two-dimensional gel electrophoresis ... For treatment with calf intestinal alkaline phosphatase (CIP), cell pellets were first lysed in RIPA as described above, followed by the addition of 10 μl of 10 U/μl CIP from New England Biolabs (Ipswich, MA) in 1× New England Biolabs buffer 2.

    Gel Extraction:

    Article Title: Mutants Resistant to LpxC Inhibitors by Rebalancing Cellular Homeostasis
    Article Snippet: The PCR fragment was purified using QIAQuick gel extraction kit (Qiagen, Valencia, CA). .. The vector was treated with calf intestinal alkaline phosphatase (New England Biolabs).

    Electrofocusing:

    Article Title: Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines
    Article Snippet: Following IEF using a Protean IEF cell (Bio-Rad) for 10,000 volt-hours at 50 μA per strip with rapid ramping, the strips were equilibrated for 20 minutes with equilibration buffer containing 6 M urea, 2% SDS, 0.05 M Tris-HCl pH 8.8, 20% glycerol, and 2% β-mercaptoethanol, followed by SDS-PAGE (10%) and Western blot analysis. .. For treatment with calf intestinal alkaline phosphatase (CIP), cell pellets were first lysed in RIPA as described above, followed by the addition of 10 μl of 10 U/μl CIP from New England Biolabs (Ipswich, MA) in 1× New England Biolabs buffer 2.

    Cross-linking Immunoprecipitation:

    Article Title: Transcriptome-wide microRNA and target dynamics in the fat body during the gonadotrophic cycle of Aedes aegypti
    Article Snippet: Paragraph title: CLIP-Seq Library Preparation. ... Beads were further digested with 1:1,000 RNase A dilution, dephosphorylated with calf intestinal alkaline phosphatase (CIP) (NEB) and ligated to 32 P-labeled 3′ RNA linker (RL3-OH) using T4 RNA ligase (Thermo Fisher Scientific).

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    New England Biolabs calf intestinal alkaline phosphatase cip
    Rdr3 is involved in accumulation of endogenous siRNAs. ( A ) Average division rate of RDR1-RDR4 ( R1-4 ) and control ( ND169 ; ICL7a ; A 51 ; GFP ) silenced cells (±standard deviation). Single cells were grown in silencing medium and individualized every day. Reduction of division rate of RDR3 silenced cells started on the fourth day of the experiment. ( B and C ) Rdr3 dependency of endogenous siRNAs. Total <t>RNA</t> was isolated on days 3, 5 and 9 of RDR3 ( R3 ) and ICL7a (control) silencing. Northern blots were probed with two adjacent 50-nt oligonucleotides corresponding to endogenous siRNAs produced from an intergenic region of scaffold 22. Probes were orientated top (B) and bottom (C), relative to transcription of the 5′-marginal ORF. The lower panels show hybridization to glutamine tRNA as a loading control. ( D ) Properties of 5′- and 3′-ends of endogenous siRNAs. Removal of 5′ phosphates with <t>CIP</t> alkaline phosphatase resulted in a ∼0.5-nt slower migration in comparison the untreated sample. Endogenous siRNAs showed sensitivity to Terminator exonuclease (Ter) and were sensitive to periodate treatment and subsequent β-elimination (P/β), indicated by ∼1.5-nt faster migration (upper blot). The second P/β-lane (right) represents the latter one with increased contrast. Controls (lower blot) were added in the same way as described for Figure 3 C. The lower panels show hybridization to glutamine tRNA as a loading control.
    Calf Intestinal Alkaline Phosphatase Cip, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/calf intestinal alkaline phosphatase cip/product/New England Biolabs
    Average 99 stars, based on 36 article reviews
    Price from $9.99 to $1999.99
    calf intestinal alkaline phosphatase cip - by Bioz Stars, 2019-10
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    80
    New England Biolabs inactive alp
    Dephosphorylation of <t>MelA2-P</t> by <t>ALP</t> increased the peptide membrane lysis potency. (A) HPLC-monitored ALP dephosphorylation of MelA2-P. The amount of MelA2-P and dephosphorylation-resulted MelA2 was monitored and traced by analytical RP-HPLC after ALP treatment for various times. The relative percentages of MelA2-P and MelA2 peptides were quantified by peak area integration and normalization. (B) Calcein leakage of the POPC vesicles after treatment with active ALP catalyzed MelA2-P and inactive ALP catalyzed MelA2-P. The inactive ALP was derived from the 100 °C heating of active ALP for 30 min and every sample had the same amount of ALP protein to avoid the influence of ALP on calcein leakage. The leakage intensity of 100 μM (lipid concentration) calcein-entrapped vesicles at a peptide-to-lipid ratio from 1/800 to 1/50 was measured for 20 min. The leakage amount reached a plateau before 20 min and the calcein fluorescence at the plateau was used as the final intensity, with n = 3.
    Inactive Alp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/inactive alp/product/New England Biolabs
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    Rdr3 is involved in accumulation of endogenous siRNAs. ( A ) Average division rate of RDR1-RDR4 ( R1-4 ) and control ( ND169 ; ICL7a ; A 51 ; GFP ) silenced cells (±standard deviation). Single cells were grown in silencing medium and individualized every day. Reduction of division rate of RDR3 silenced cells started on the fourth day of the experiment. ( B and C ) Rdr3 dependency of endogenous siRNAs. Total RNA was isolated on days 3, 5 and 9 of RDR3 ( R3 ) and ICL7a (control) silencing. Northern blots were probed with two adjacent 50-nt oligonucleotides corresponding to endogenous siRNAs produced from an intergenic region of scaffold 22. Probes were orientated top (B) and bottom (C), relative to transcription of the 5′-marginal ORF. The lower panels show hybridization to glutamine tRNA as a loading control. ( D ) Properties of 5′- and 3′-ends of endogenous siRNAs. Removal of 5′ phosphates with CIP alkaline phosphatase resulted in a ∼0.5-nt slower migration in comparison the untreated sample. Endogenous siRNAs showed sensitivity to Terminator exonuclease (Ter) and were sensitive to periodate treatment and subsequent β-elimination (P/β), indicated by ∼1.5-nt faster migration (upper blot). The second P/β-lane (right) represents the latter one with increased contrast. Controls (lower blot) were added in the same way as described for Figure 3 C. The lower panels show hybridization to glutamine tRNA as a loading control.

    Journal: Nucleic Acids Research

    Article Title: Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia

    doi: 10.1093/nar/gkq131

    Figure Lengend Snippet: Rdr3 is involved in accumulation of endogenous siRNAs. ( A ) Average division rate of RDR1-RDR4 ( R1-4 ) and control ( ND169 ; ICL7a ; A 51 ; GFP ) silenced cells (±standard deviation). Single cells were grown in silencing medium and individualized every day. Reduction of division rate of RDR3 silenced cells started on the fourth day of the experiment. ( B and C ) Rdr3 dependency of endogenous siRNAs. Total RNA was isolated on days 3, 5 and 9 of RDR3 ( R3 ) and ICL7a (control) silencing. Northern blots were probed with two adjacent 50-nt oligonucleotides corresponding to endogenous siRNAs produced from an intergenic region of scaffold 22. Probes were orientated top (B) and bottom (C), relative to transcription of the 5′-marginal ORF. The lower panels show hybridization to glutamine tRNA as a loading control. ( D ) Properties of 5′- and 3′-ends of endogenous siRNAs. Removal of 5′ phosphates with CIP alkaline phosphatase resulted in a ∼0.5-nt slower migration in comparison the untreated sample. Endogenous siRNAs showed sensitivity to Terminator exonuclease (Ter) and were sensitive to periodate treatment and subsequent β-elimination (P/β), indicated by ∼1.5-nt faster migration (upper blot). The second P/β-lane (right) represents the latter one with increased contrast. Controls (lower blot) were added in the same way as described for Figure 3 C. The lower panels show hybridization to glutamine tRNA as a loading control.

    Article Snippet: Removal of 5′ phosphates was carried out by treating 20 µg of total RNA with 5 U calf intestinal alkaline phosphatase (CIP) (New England Biolabs, Frankfurt am Main, Germany) for 1 h at 37°C.

    Techniques: Standard Deviation, Isolation, Northern Blot, Produced, Hybridization, Migration

    Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single oligo in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total RNA with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.

    Journal: Nucleic Acids Research

    Article Title: Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia

    doi: 10.1093/nar/gkq131

    Figure Lengend Snippet: Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single oligo in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total RNA with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.

    Article Snippet: Removal of 5′ phosphates was carried out by treating 20 µg of total RNA with 5 U calf intestinal alkaline phosphatase (CIP) (New England Biolabs, Frankfurt am Main, Germany) for 1 h at 37°C.

    Techniques: Sequencing, Migration, Modification, Hybridization

    Scheme of the pocket-sized RNA-seq method. 5P-, 5′-phosphate group; 3OH-, 3′-hydroxyl group; 5-, dephosphorylated 5′-end; (AAA)n-3OH, polyadenylated 3′-end; (AAA)n and (TTT)n, polyA and polyT sequences, respectively; RACE, Rapid amplification of cDNA ends; RT, reverse transcription; CIP, calf intestinal phosphatase; oligo-biot, oligonucleotide biotinylated in 5′-end.

    Journal: Non-Coding RNA

    Article Title: “Pocket-sized RNA-Seq”: A Method to Capture New Mature microRNA Produced from a Genomic Region of Interest

    doi: 10.3390/ncrna1020127

    Figure Lengend Snippet: Scheme of the pocket-sized RNA-seq method. 5P-, 5′-phosphate group; 3OH-, 3′-hydroxyl group; 5-, dephosphorylated 5′-end; (AAA)n-3OH, polyadenylated 3′-end; (AAA)n and (TTT)n, polyA and polyT sequences, respectively; RACE, Rapid amplification of cDNA ends; RT, reverse transcription; CIP, calf intestinal phosphatase; oligo-biot, oligonucleotide biotinylated in 5′-end.

    Article Snippet: One microgram of bait RNA was therefore dephosphorylated using 1 U CIP (Alkaline Phosphatase, Calf Intestinal, New England Biolabs, Evry, France) at 37 °C for 60 min and RNAs were purified by phenol-chloroform extraction.

    Techniques: RNA Sequencing Assay, Rapid Amplification of cDNA Ends

    Western blot analysis of the 43-kDa gap junction protein connexin 43 (Cx43) from the hearts of two control rats lysed in either +PI or −PI buffer. Lysate in the −PI buffer was further treated with calf intestinal alkaline phosphatase (CIP).

    Journal:

    Article Title: Hindlimb unloading results in increased predisposition to cardiac arrhythmias and alters left ventricular connexin 43 expression

    doi: 10.1152/ajpregu.00391.2012

    Figure Lengend Snippet: Western blot analysis of the 43-kDa gap junction protein connexin 43 (Cx43) from the hearts of two control rats lysed in either +PI or −PI buffer. Lysate in the −PI buffer was further treated with calf intestinal alkaline phosphatase (CIP).

    Article Snippet: To determine the specificity of the monoclonal antibody to detect unphosphorylated Cx43, −PI-extracted samples were treated with 2 μl of calf intestinal alkaline phosphatase (+CIP) (New England BioLabs).

    Techniques: Western Blot

    Dephosphorylation of MelA2-P by ALP increased the peptide membrane lysis potency. (A) HPLC-monitored ALP dephosphorylation of MelA2-P. The amount of MelA2-P and dephosphorylation-resulted MelA2 was monitored and traced by analytical RP-HPLC after ALP treatment for various times. The relative percentages of MelA2-P and MelA2 peptides were quantified by peak area integration and normalization. (B) Calcein leakage of the POPC vesicles after treatment with active ALP catalyzed MelA2-P and inactive ALP catalyzed MelA2-P. The inactive ALP was derived from the 100 °C heating of active ALP for 30 min and every sample had the same amount of ALP protein to avoid the influence of ALP on calcein leakage. The leakage intensity of 100 μM (lipid concentration) calcein-entrapped vesicles at a peptide-to-lipid ratio from 1/800 to 1/50 was measured for 20 min. The leakage amount reached a plateau before 20 min and the calcein fluorescence at the plateau was used as the final intensity, with n = 3.

    Journal: Chemical Science

    Article Title: Selective inhibition of cancer cells by enzyme-induced gain of function of phosphorylated melittin analogues †Electronic supplementary information (ESI) available: Mass spectra and other materials. See DOI: 10.1039/c7sc03217j

    doi: 10.1039/c7sc03217j

    Figure Lengend Snippet: Dephosphorylation of MelA2-P by ALP increased the peptide membrane lysis potency. (A) HPLC-monitored ALP dephosphorylation of MelA2-P. The amount of MelA2-P and dephosphorylation-resulted MelA2 was monitored and traced by analytical RP-HPLC after ALP treatment for various times. The relative percentages of MelA2-P and MelA2 peptides were quantified by peak area integration and normalization. (B) Calcein leakage of the POPC vesicles after treatment with active ALP catalyzed MelA2-P and inactive ALP catalyzed MelA2-P. The inactive ALP was derived from the 100 °C heating of active ALP for 30 min and every sample had the same amount of ALP protein to avoid the influence of ALP on calcein leakage. The leakage intensity of 100 μM (lipid concentration) calcein-entrapped vesicles at a peptide-to-lipid ratio from 1/800 to 1/50 was measured for 20 min. The leakage amount reached a plateau before 20 min and the calcein fluorescence at the plateau was used as the final intensity, with n = 3.

    Article Snippet: For the calcein leakage experiment, MelA2-P (100 μM) was incubated with 100 U ml–1 active and inactive ALP (Alkaline Phosphatase, Calf Intestinal, New England Biolabs) in Tris buffer (10 mM Tris, 150 mM NaCl, pH 7.4) at 37 °C for 5 h and then all samples were heated at 100 °C for 30 min to stop catalytic reactions.

    Techniques: De-Phosphorylation Assay, ALP Assay, Lysis, High Performance Liquid Chromatography, Derivative Assay, Concentration Assay, Fluorescence

    MelA2-P selectively kills cancer cells with high ALP activity. (A) The relative ALP activity in MDCK, Hela and Saos-2. (B) The cell viability of MDCK, Hela, and Saos-2 cells after treatment with 1.5 μM MelA2 and MelA2-P for 24 h. The cell viability of (C) MDCK, (D) Hela and (E) Saos-2 cells after treatment with different concentrations of MelA2-P and MelA2 for 24 h. (F) The cell viability of Saos-2 after treatment with the ALP inhibitor levamisole and peptides (MelA2 or MelA2-P). The cells in black histograms were not treated by levamisole, and the cells in white histograms were incubated with 0.5 mM levamisole for 3 h before the addition of the mixture of 1 μM peptides (MelA2 or MelA2-P) and 0.5 mM levamisole for 24 h of culturing. For the control group, the peptide treatments were replaced by equivalent buffer. n = 3, * p

    Journal: Chemical Science

    Article Title: Selective inhibition of cancer cells by enzyme-induced gain of function of phosphorylated melittin analogues †Electronic supplementary information (ESI) available: Mass spectra and other materials. See DOI: 10.1039/c7sc03217j

    doi: 10.1039/c7sc03217j

    Figure Lengend Snippet: MelA2-P selectively kills cancer cells with high ALP activity. (A) The relative ALP activity in MDCK, Hela and Saos-2. (B) The cell viability of MDCK, Hela, and Saos-2 cells after treatment with 1.5 μM MelA2 and MelA2-P for 24 h. The cell viability of (C) MDCK, (D) Hela and (E) Saos-2 cells after treatment with different concentrations of MelA2-P and MelA2 for 24 h. (F) The cell viability of Saos-2 after treatment with the ALP inhibitor levamisole and peptides (MelA2 or MelA2-P). The cells in black histograms were not treated by levamisole, and the cells in white histograms were incubated with 0.5 mM levamisole for 3 h before the addition of the mixture of 1 μM peptides (MelA2 or MelA2-P) and 0.5 mM levamisole for 24 h of culturing. For the control group, the peptide treatments were replaced by equivalent buffer. n = 3, * p

    Article Snippet: For the calcein leakage experiment, MelA2-P (100 μM) was incubated with 100 U ml–1 active and inactive ALP (Alkaline Phosphatase, Calf Intestinal, New England Biolabs) in Tris buffer (10 mM Tris, 150 mM NaCl, pH 7.4) at 37 °C for 5 h and then all samples were heated at 100 °C for 30 min to stop catalytic reactions.

    Techniques: ALP Assay, Activity Assay, Incubation