10x antarctic phosphatase reaction buffer  (New England Biolabs)


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    Name:
    Antarctic Phosphatase Reaction Buffer
    Description:

    Catalog Number:
    B0289S
    Price:
    None
    Score:
    85
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    Structured Review

    New England Biolabs 10x antarctic phosphatase reaction buffer

    https://www.bioz.com/result/10x antarctic phosphatase reaction buffer/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    10x antarctic phosphatase reaction buffer - by Bioz Stars, 2019-10
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    Related Articles

    Centrifugation:

    Article Title: Calcium-dependent protein kinases play an essential role in a plant defence response
    Article Snippet: After addition of 50 µl of protein G and a further incubation for 45 min, samples were harvested by centrifugation and washed with solubilization buffer containing 0.3% Triton X-100 (first and second wash), 0.3% Triton X-100 and 1 M NaCl (third wash). .. For in vitro interconversion experiments, reactions with NtCDPK2-myc immunoprecipitates/beads were washed once with phosphatase buffer (NEB) and resuspended in 100 µl of the same buffer containing 2 U of non-specific λ phosphatase (NEB) in the absence or presence (control) of 50 mM NaF and 10 mM Na3 VO4 .

    Article Title: The Hem protein mediates neuronal migration by inhibiting WAVE degradation and functions opposite of Abelson tyrosine kinase
    Article Snippet: Twenty embryos were collected, dechorionated and homogenized in 37.5μL of the Lysis buffer (see above). .. After centrifugation, supernatant (~37.5μL) was collected and incubated with Lambda protein phosphatase (Lambda PP, 100U) in NEB phosphatase buffer (5μL of 10X) and MnCl2 (5μL of 10 mM solution) in a total final volume of 50μL for 30 min at 30°C. .. For the control, lambda PP was omitted.

    Phosphatase Assay:

    Article Title: BRCA1 Inhibits Membrane Estrogen and Growth Factor Receptor Signaling to Cell Proliferation in Breast Cancer
    Article Snippet: Paragraph title: Phosphatase activity assay. ... The cells were washed twice with Dulbecco's minimal essential medium, scraped, and then sonicated in phosphatase activity buffer (New England Biolabs).

    Article Title: Phosphatase 1 Nuclear Targeting Subunit Is an Essential Regulator of M-phase Entry, Maintenance, and Exit
    Article Snippet: Following the kinase reaction, the protein bound to beads was washed with modified extract buffer (1 m KCl, 11 m m MgCl2 , 100 m m HEPES, pH 7.7, 500 m m sucrose, and 5 m m EGTA, pH 7.7) and eluted with 10 m m maltose in modified extract buffer. .. For the PP1 phosphatase assay, prephosphorylated histone H3 peptide was incubated with PP1 (New England Biolabs) in phosphatase buffer (New England Biolabs) with and without Pnuts protein at room temperature for the time indicated. .. Small aliquots were removed at the indicated time points and diluted 1:10 in Laemmli sample buffer, resolved by SDS-PAGE, and detected by immunoblotting.

    Synthesized:

    Article Title: mRNA Transfection of Mouse and Human Neural Stem Cell Cultures
    Article Snippet: Capped single stranded RNA was synthesized via IVT using the MEGAscript-T7 kit (Life Technologies). .. To remove 5′ triphosphate moieties from uncapped transcripts, 10 µl of Antarctic Phosphatase reaction buffer and 5 µl (25U) of Antarctic Phosphatase (New England BioLabs) was added to each RNA product and incubated for 30-minutes at 37°C.

    Construct:

    Article Title: Comparison of CAGE and RNA-seq transcriptome profiling using clonally amplified and single-molecule next-generation sequencing
    Article Snippet: We constructed sequencing libraries starting from 500 ng of total RNA. .. Purified RNA was dephosphorylated by adding 2 μL of 10× phosphatase buffer, 5 units of Antarctic phosphatase (NEB), and 40 units of RNaseOUT (Invitrogen) and incubating for 30 min at 37°C followed by 5 min at 65°C.

    SDS-Gel:

    Article Title: The Hem protein mediates neuronal migration by inhibiting WAVE degradation and functions opposite of Abelson tyrosine kinase
    Article Snippet: After centrifugation, supernatant (~37.5μL) was collected and incubated with Lambda protein phosphatase (Lambda PP, 100U) in NEB phosphatase buffer (5μL of 10X) and MnCl2 (5μL of 10 mM solution) in a total final volume of 50μL for 30 min at 30°C. .. After centrifugation, supernatant (~37.5μL) was collected and incubated with Lambda protein phosphatase (Lambda PP, 100U) in NEB phosphatase buffer (5μL of 10X) and MnCl2 (5μL of 10 mM solution) in a total final volume of 50μL for 30 min at 30°C.

    Incubation:

    Article Title: Borrelia burgdorferi bb0426 encodes a 2?-deoxyribosyltransferase that plays a central role in purine salvage
    Article Snippet: A 50 μl aliquot of the DNA preparation was denatured (97°C for 2 min) and then immediately placed on ice. .. Ten microliters of 0.3M NaOAc pH 5.3, 5 μl 20 mM ZnSO4 and 10 μl P1 Nuclease (Sigma-Aldrich) were added to each sample and incubated at 37°C for 2 h. Next, 10 μl of 10× Antarctic phosphatase buffer and 2 μl (10 units) of Antarctic phosphatase (NEB, Ipswich, MA) were added and incubated at 37°C for 20 h. The reaction mixture was analysed by HPLC under the following conditions: 75 μl aliquot of sample was injected onto a Supelcosil LC-18-S 4.6 mm × 150 mm, 5 μm analytical column (Supelco) equilibrated with 50 mM potassium phosphate, pH 4.5, containing 8% methanol. .. Furthermore, spectral data were collected for each peak using a diode array detector scanning from 200 to 600 nm.

    Article Title: Calcium-dependent protein kinases play an essential role in a plant defence response
    Article Snippet: After addition of 50 µl of protein G and a further incubation for 45 min, samples were harvested by centrifugation and washed with solubilization buffer containing 0.3% Triton X-100 (first and second wash), 0.3% Triton X-100 and 1 M NaCl (third wash). .. For in vitro interconversion experiments, reactions with NtCDPK2-myc immunoprecipitates/beads were washed once with phosphatase buffer (NEB) and resuspended in 100 µl of the same buffer containing 2 U of non-specific λ phosphatase (NEB) in the absence or presence (control) of 50 mM NaF and 10 mM Na3 VO4 .

    Article Title: Orm family proteins mediate sphingolipid homeostasis
    Article Snippet: The anti-Flag resin was then washed 2× 1ml with Tween IP buffer, 1× 1ml with Tween/Urea wash buffer (100mM Tris-Cl, pH 7.8, 100mM NaCl, 0.5% Tween-20, 2M Urea), and 2× 1ml in phosphatase buffer (NEBuffer 3; New England Biolabs), with 0.1% Tween-20). .. The washed resin was then resuspended in 54µl phosphatase buffer + 6µl calf intestine phosphatase (New England Biolabs) and incubated for 1 hr at 37°C. .. After phosphatase treatment, the resin was sequentially washed with 1ml Tween IP buffer and 1ml in SDS/DOC wash buffer (50mM Tris-Cl, pH 7.8, 0.1% SDS, 0.1% Na-deoxycholate).

    Article Title: Phosphorylation regulates human polη stability and damage bypass throughout the cell cycle
    Article Snippet: Flag-HA Polη was purified and analyzed by liquid chromatography-tandem mass spectrometry (MS) as described in ( ). .. A total of 120 μg protein extracts were incubated in phosphatase buffer (New England Biolabs) with or without 1600U of λ phosphatase (New England Biolabs) for 30 min at 30°C. .. After incubation the reaction was diluted with rehydration buffer up to a volume of 200 μl and processed as previously described for 2D-PAGE.

    Article Title: Comparison of CAGE and RNA-seq transcriptome profiling using clonally amplified and single-molecule next-generation sequencing
    Article Snippet: Purified RNA was dephosphorylated by adding 2 μL of 10× phosphatase buffer, 5 units of Antarctic phosphatase (NEB), and 40 units of RNaseOUT (Invitrogen) and incubating for 30 min at 37°C followed by 5 min at 65°C. .. Purified RNA was dephosphorylated by adding 2 μL of 10× phosphatase buffer, 5 units of Antarctic phosphatase (NEB), and 40 units of RNaseOUT (Invitrogen) and incubating for 30 min at 37°C followed by 5 min at 65°C.

    Article Title: BRCA1 Inhibits Membrane Estrogen and Growth Factor Receptor Signaling to Cell Proliferation in Breast Cancer
    Article Snippet: The cells were washed twice with Dulbecco's minimal essential medium, scraped, and then sonicated in phosphatase activity buffer (New England Biolabs). .. After centrifugation, the supernatants (100 μl) from the cells subjected to each treatment were added in separate tubes to equal aliquots of 32 P-labeled ERK.

    Article Title: TBK1 Provides Context-Selective Support of the Activated AKT/mTOR Pathway in Lung Cancer
    Article Snippet: Samples were then heated to ≥ 95C for ≥ 5 min in SDS sample buffer (+β-me) and then separated by SDS-PAGE, followed by immunoblotting. .. Recombinant human S6K (Abnova) was first incubated with lambda protein phosphatase in phosphatase buffer (NEB) at a ratio of 3 units of phosphatase per 10ng of protein for 60 min at 30C. .. Following phosphatase treatment, 100ng of S6K was incubated with or without 50ng recombinant active TBK1 (EMD Millipore) in kinase buffer containing phosphatase inhibitors (25 mM Tris-HCl pH 7.4, 10mM MgCl2 , 0.1mM Na3 VO4 , 2mM DTT, 5mM β-glycerosphosphate) in the presence or absence of 200µM ATP for 30 min at 30C (Total reaction volume = 50µL).

    Article Title: The Hem protein mediates neuronal migration by inhibiting WAVE degradation and functions opposite of Abelson tyrosine kinase
    Article Snippet: Twenty embryos were collected, dechorionated and homogenized in 37.5μL of the Lysis buffer (see above). .. After centrifugation, supernatant (~37.5μL) was collected and incubated with Lambda protein phosphatase (Lambda PP, 100U) in NEB phosphatase buffer (5μL of 10X) and MnCl2 (5μL of 10 mM solution) in a total final volume of 50μL for 30 min at 30°C. .. For the control, lambda PP was omitted.

    Article Title: Regulation of the filament structure and assembly of Acanthamoeba myosin II by phosphorylation of serines in the heavy-chain nonhelical tailpiece
    Article Snippet: A 100-µL aliquot was incubated with 5 µL of 10-fold–diluted kinase preparation at 30 °C in a shaker for 1 h. Then 0.6 mg/mL of the resultant pWT was dissolved in a buffer of 1 mM MgCl2 , 1 mM ATP, 2.5 mM KCl, and 10 mM imidazole (pH 7.0). .. An 40-µL aliquot was mixed with 5 µL of 10× concentrated phosphatase buffer (New England BioLabs) and 5 µL of 10× concentrated MnCl2 solution (New England BioLabs) and was incubated with 1 µL of lambda protein phosphatase (New England BioLabs) for 10 min at room temperature. .. For electron microscopy, myosins were dialyzed against 10 mM imidazole (pH 7.0) and 25 mM KCl overnight and then were mixed with ATP and MgCl2 to a final concentration of 120 nM myosin, 10 mM imidazole (pH 7.0), 1 mM ATP, 2.5 mM KCl, and MgCl2 as indicated.

    Article Title: Feeder-Free Derivation of Human Induced Pluripotent Stem Cells with Messenger RNA
    Article Snippet: Reactions were incubated 4–6 hours at 37°C and treated with 2 uL TURBO DNase for a further 15 minutes at 37°C before being purified on MEGAclear (Ambion) spin columns, the RNA products being eluted in a volume of 100 uL. .. To remove immunogenic 5′ triphosphate moieties from uncapped transcripts, 10 uL of Antarctic Phosphatase reaction buffer and 3 uL of Antarctic Phosphatase (NEB) was added to each prep.

    Article Title: mRNA Transfection of Mouse and Human Neural Stem Cell Cultures
    Article Snippet: RNA products were eluted with 85 µl of H2 O in each column. .. To remove 5′ triphosphate moieties from uncapped transcripts, 10 µl of Antarctic Phosphatase reaction buffer and 5 µl (25U) of Antarctic Phosphatase (New England BioLabs) was added to each RNA product and incubated for 30-minutes at 37°C. .. Synthesized RNA products were then repurified using RNeasy Mini Columns (Qiagen) and quantitated by spectrophotometry and denaturing gel electrophoresis.

    Article Title: Aire controls gene expression in the thymic epithelium with ordered stochasticity
    Article Snippet: Samples were immediately incubated at 94°C (lid 105°C) for 2 minutes, then immediately transferred onto ice and the reaction was stopped by adding quickly 2µL of 10X RNA Fragmentation Stop Solution. .. Then the RNA was 3’ dephosphorylated and 5’ phosphorylated by adding 30µL of a mix containing 21.5µL of RNAse-free water, 5µL of 10X Antarctic Phosphatase Reaction Buffer (NEB M0289), 0.5µL of ATP (100mM stock, ATP Tris buffered Thermo Scientific #R1441), 1µL of RNAseOut and 2µL of T4 PolyNucleotide Kinase (10U/µL, NEB M0201S), and incubating at 37°C for 1 hour.

    Article Title: Phosphatase 1 Nuclear Targeting Subunit Is an Essential Regulator of M-phase Entry, Maintenance, and Exit
    Article Snippet: Following the kinase reaction, the protein bound to beads was washed with modified extract buffer (1 m KCl, 11 m m MgCl2 , 100 m m HEPES, pH 7.7, 500 m m sucrose, and 5 m m EGTA, pH 7.7) and eluted with 10 m m maltose in modified extract buffer. .. For the PP1 phosphatase assay, prephosphorylated histone H3 peptide was incubated with PP1 (New England Biolabs) in phosphatase buffer (New England Biolabs) with and without Pnuts protein at room temperature for the time indicated. .. Small aliquots were removed at the indicated time points and diluted 1:10 in Laemmli sample buffer, resolved by SDS-PAGE, and detected by immunoblotting.

    Article Title: RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases
    Article Snippet: Beads were washed twice in T1 buffer and phosphatase reaction buffer (50 mM Tris-HCl pH 7.0, 1 mM MgCl2 , 0.1 mM ZnCl2 ). .. For dephosphorylation, 1× antarctic phosphatase reaction buffer (NEB, Germany) with 1 U/µL of antarctic phosphatase and 1 U/µL of RNase OUT (Invitrogen) were added and the suspension was incubated at 37°C for 30 min and 800 rpm. .. Beads were washed once in phosphatase wash buffer (50 mM Tris-HCl pH 7.5, 20 mM EGTA, 0.5% NP-40) and twice in polynucleotide kinase (PNK) buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM MgCl2 ).

    Activity Assay:

    Article Title: Calcium-dependent protein kinases play an essential role in a plant defence response
    Article Snippet: To determine kinase activity of transiently expressed NtCDPK2-myc, 150 µg of solubilized crude membrane extracts were incubated with 300 µl of solubilization buffer (lacking the detergent and DTT) and 0.25 µg of monoclonal anti-c-myc antibody with end-over-end rotation for 90 min at 6°C. .. For in vitro interconversion experiments, reactions with NtCDPK2-myc immunoprecipitates/beads were washed once with phosphatase buffer (NEB) and resuspended in 100 µl of the same buffer containing 2 U of non-specific λ phosphatase (NEB) in the absence or presence (control) of 50 mM NaF and 10 mM Na3 VO4 .

    Article Title: BRCA1 Inhibits Membrane Estrogen and Growth Factor Receptor Signaling to Cell Proliferation in Breast Cancer
    Article Snippet: Two days earlier, a second set of MCF-7 cells had been transfected with pcDNA3 (control) or wt BRCA1 and then recovered and synchronized. .. The cells were washed twice with Dulbecco's minimal essential medium, scraped, and then sonicated in phosphatase activity buffer (New England Biolabs). .. After centrifugation, the supernatants (100 μl) from the cells subjected to each treatment were added in separate tubes to equal aliquots of 32 P-labeled ERK.

    Caspase Assay:

    Article Title:
    Article Snippet: Cells were treated for 45 min with 1 μm okadaic acid (Bioshop Canada Inc., Burlington, Ontario, Canada) and then lysed in caspase assay buffer (0.1% CHAPS, 20 mm PIPES (pH 7.4), 100 mm NaCl) and protease and phosphatase inhibitors (leupeptin (10 μg/ml), 0.1 mm phenylmethylsulfonyl fluoride (PMSF), pepstatin A (10 μg/ml), aprotinin (5 μg/ml), 50 mm NaF, 1 μm microcystin, and 1 mm sodium orthovanadate). .. After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C.

    Expressing:

    Article Title: Cyanobacterial circadian clockwork: roles of KaiA, KaiB and the kaiBC promoter in regulating KaiC
    Article Snippet: For phosphatase treatment of cyanobacterial extracts, the concentrated extracts (10 µg/µl protein) from the IPTG-induced Δ kaiC strain expressing trc p:: kaiC were prepared in the following buffer: 50 mM HEPES pH 7.4, 130 mM KCl, 5% glycerol, 2.5 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml pepstatin and 5 mM phenylmethylsulfonyl fluoride. .. About 1 µg of purified KaiC::His6 or 30 µg of total protein from extracts was diluted in 1× phosphatase buffer (New England Biolabs, Beverly, MA).

    Article Title: Parallel Regulation of von Hippel-Lindau Disease by pVHL-Mediated Degradation of B-Myb and Hypoxia-Inducible Factor α
    Article Snippet: 786-O cells (2.6 × 106 ) expressing 3×HA–B-Myb (wild type [WT]or Y15A mutant) or control cells were cultured in either one 10-cm dish (confluent condition) or two 15-cm dishes (sparse condition) for 2 days with one change of culture medium. .. Phosphatase treatment was performed as follows: 3×HA–B-Myb was immunoprecipitated and washed with phosphatase buffer supplied by New England BioLabs (NEB; Tokyo, Japan).

    Bradford Assay:

    Article Title:
    Article Snippet: After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C. .. After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C.

    Modification:

    Article Title: Feeder-Free Derivation of Human Induced Pluripotent Stem Cells with Messenger RNA
    Article Snippet: Cap analog and modified NTPs were purchased from Trilink Biotechnologies. .. To remove immunogenic 5′ triphosphate moieties from uncapped transcripts, 10 uL of Antarctic Phosphatase reaction buffer and 3 uL of Antarctic Phosphatase (NEB) was added to each prep.

    Article Title:
    Article Snippet: HeLa cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml). .. After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C.

    Article Title: Phosphatase 1 Nuclear Targeting Subunit Is an Essential Regulator of M-phase Entry, Maintenance, and Exit
    Article Snippet: Following the kinase reaction, the protein bound to beads was washed with modified extract buffer (1 m KCl, 11 m m MgCl2 , 100 m m HEPES, pH 7.7, 500 m m sucrose, and 5 m m EGTA, pH 7.7) and eluted with 10 m m maltose in modified extract buffer. .. For the PP1 phosphatase assay, prephosphorylated histone H3 peptide was incubated with PP1 (New England Biolabs) in phosphatase buffer (New England Biolabs) with and without Pnuts protein at room temperature for the time indicated.

    Western Blot:

    Article Title: The Hem protein mediates neuronal migration by inhibiting WAVE degradation and functions opposite of Abelson tyrosine kinase
    Article Snippet: After centrifugation, supernatant (~37.5μL) was collected and incubated with Lambda protein phosphatase (Lambda PP, 100U) in NEB phosphatase buffer (5μL of 10X) and MnCl2 (5μL of 10 mM solution) in a total final volume of 50μL for 30 min at 30°C. .. After centrifugation, supernatant (~37.5μL) was collected and incubated with Lambda protein phosphatase (Lambda PP, 100U) in NEB phosphatase buffer (5μL of 10X) and MnCl2 (5μL of 10 mM solution) in a total final volume of 50μL for 30 min at 30°C.

    Article Title:
    Article Snippet: Cells were lysed by sonication with two pulses of 5 s each, and the samples were subsequently cleared by ultracentrifugation at 13,000 × g if being used for Western blot analysis or 140,000 × g if being used for proteomic analysis. .. After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C.

    Article Title: A second Wpl1 anti‐cohesion pathway requires dephosphorylation of fission yeast kleisin Rad21 by PP4
    Article Snippet: Protein extracts, IPs and Western blotting were done as described (Feytout et al , ). .. Rad21‐9PK bound to magnetic beads was washed twice in phosphatase buffer (50 mM HEPES; 100 mM NaCl; 2 mM DTT; 0.01% Brij 35 pH 7.5) and beads dispensed into three 50‐μl aliquots (without phosphatase, 400 units phosphatase (New England Biolabs), 400 units phosphatase and 50 mM Na‐vanadate and 10 mM β‐glycerophosphate).

    Kinase Assay:

    Article Title: TBK1 Provides Context-Selective Support of the Activated AKT/mTOR Pathway in Lung Cancer
    Article Snippet: Paragraph title: In Vitro Kinase Assay ... Recombinant human S6K (Abnova) was first incubated with lambda protein phosphatase in phosphatase buffer (NEB) at a ratio of 3 units of phosphatase per 10ng of protein for 60 min at 30C.

    High Performance Liquid Chromatography:

    Article Title: Borrelia burgdorferi bb0426 encodes a 2?-deoxyribosyltransferase that plays a central role in purine salvage
    Article Snippet: A 50 μl aliquot of the DNA preparation was denatured (97°C for 2 min) and then immediately placed on ice. .. Ten microliters of 0.3M NaOAc pH 5.3, 5 μl 20 mM ZnSO4 and 10 μl P1 Nuclease (Sigma-Aldrich) were added to each sample and incubated at 37°C for 2 h. Next, 10 μl of 10× Antarctic phosphatase buffer and 2 μl (10 units) of Antarctic phosphatase (NEB, Ipswich, MA) were added and incubated at 37°C for 20 h. The reaction mixture was analysed by HPLC under the following conditions: 75 μl aliquot of sample was injected onto a Supelcosil LC-18-S 4.6 mm × 150 mm, 5 μm analytical column (Supelco) equilibrated with 50 mM potassium phosphate, pH 4.5, containing 8% methanol. .. Furthermore, spectral data were collected for each peak using a diode array detector scanning from 200 to 600 nm.

    Transfection:

    Article Title: BRCA1 Inhibits Membrane Estrogen and Growth Factor Receptor Signaling to Cell Proliferation in Breast Cancer
    Article Snippet: Two days earlier, a second set of MCF-7 cells had been transfected with pcDNA3 (control) or wt BRCA1 and then recovered and synchronized. .. The cells were washed twice with Dulbecco's minimal essential medium, scraped, and then sonicated in phosphatase activity buffer (New England Biolabs).

    Ligation:

    Article Title: Comparison of CAGE and RNA-seq transcriptome profiling using clonally amplified and single-molecule next-generation sequencing
    Article Snippet: Purified RNA was dephosphorylated by adding 2 μL of 10× phosphatase buffer, 5 units of Antarctic phosphatase (NEB), and 40 units of RNaseOUT (Invitrogen) and incubating for 30 min at 37°C followed by 5 min at 65°C. .. A mixture of 2 μM preadenylated 3′ DNA adaptor and 1 μL concentrated RNA was incubated for 2 min at 70°C and immediately kept on ice for 2 min. One microliter of 10× T4 RNA ligase 2 truncated buffer, 0.8 μL of 100 mM MgCl2 , 20 units of RNaseOUT, and 200 units of RNA ligase 2 truncated (NEB) were added to make a 10 μL reaction.

    Buffer Exchange:

    Article Title:
    Article Snippet: The protein concentration was determined by using the Bradford assay. .. After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C. .. Next, lysates were treated with 50, 500, or 5000 nm of caspase-3 and caspase-7 for 1 h at 37 °C, and the reaction was terminated by the addition of 6 μm irreversible caspase inhibitor z-VAD-fmk (Sigma).

    Cell Culture:

    Article Title: Parallel Regulation of von Hippel-Lindau Disease by pVHL-Mediated Degradation of B-Myb and Hypoxia-Inducible Factor α
    Article Snippet: 786-O cells (2.6 × 106 ) expressing 3×HA–B-Myb (wild type [WT]or Y15A mutant) or control cells were cultured in either one 10-cm dish (confluent condition) or two 15-cm dishes (sparse condition) for 2 days with one change of culture medium. .. Phosphatase treatment was performed as follows: 3×HA–B-Myb was immunoprecipitated and washed with phosphatase buffer supplied by New England BioLabs (NEB; Tokyo, Japan).

    Article Title:
    Article Snippet: HeLa cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml). .. After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C.

    SDS Page:

    Article Title: Calcium-dependent protein kinases play an essential role in a plant defence response
    Article Snippet: For in vitro interconversion experiments, reactions with NtCDPK2-myc immunoprecipitates/beads were washed once with phosphatase buffer (NEB) and resuspended in 100 µl of the same buffer containing 2 U of non-specific λ phosphatase (NEB) in the absence or presence (control) of 50 mM NaF and 10 mM Na3 VO4 . .. After incubation for 5 min at 30°C, 20 µl of supernatant were spotted on P81 phosphocellulose paper squares and the incorporatation of phosphate was determined as described ( ).

    Article Title: Cyanobacterial circadian clockwork: roles of KaiA, KaiB and the kaiBC promoter in regulating KaiC
    Article Snippet: About 1 µg of purified KaiC::His6 or 30 µg of total protein from extracts was diluted in 1× phosphatase buffer (New England Biolabs, Beverly, MA). .. For the samples that included phosphatase, 1000 U of lambda protein phosphatase (λPPase; New England Biolabs) were added, and the reaction was incubated at 30°C for 1 h in the presence or absence of 40 mM sodium vanadate.

    Article Title: TBK1 Provides Context-Selective Support of the Activated AKT/mTOR Pathway in Lung Cancer
    Article Snippet: Recombinant human S6K (Abnova) was first incubated with lambda protein phosphatase in phosphatase buffer (NEB) at a ratio of 3 units of phosphatase per 10ng of protein for 60 min at 30C. .. Recombinant human S6K (Abnova) was first incubated with lambda protein phosphatase in phosphatase buffer (NEB) at a ratio of 3 units of phosphatase per 10ng of protein for 60 min at 30C.

    Generated:

    Article Title: Improving the performance of true single molecule sequencing for ancient DNA
    Article Snippet: For each spiking experiment, we further assumed a binomial distribution with Rmin as the probability of success for estimating the number of expected endogenous sequences given the total number of sequences generated. .. For the phosphatase reactions, 0.5 μl of the DNA extracts were mixed with 8 μl of nuclease-free water, 1 μl of NEB Antarctic Phosphatase 10X Reaction buffer and 2.5 units of NEB Antarctic Phosphatase.

    Inhibition:

    Article Title: Parallel Regulation of von Hippel-Lindau Disease by pVHL-Mediated Degradation of B-Myb and Hypoxia-Inducible Factor α
    Article Snippet: For tyrosine kinase inhibition, the following inhibitors were added to the culture medium for 1 day before harvesting the cells: 0.1 μM epidermal growth factor receptor (EGFR) inhibitor PD153035, 5 μM ErbB2 inhibitor AG825, 0.1 μM insulin-like growth factor receptor (IGFR) inhibitor picropodophyllin, 1 μM mesenchymal epithelial transition factor (MET) inhibitor SU11274, and 5 μM vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) inhibitor SU4312. .. Phosphatase treatment was performed as follows: 3×HA–B-Myb was immunoprecipitated and washed with phosphatase buffer supplied by New England BioLabs (NEB; Tokyo, Japan).

    Protein Concentration:

    Article Title:
    Article Snippet: After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C. .. After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C.

    Sequencing:

    Article Title: Comparison of CAGE and RNA-seq transcriptome profiling using clonally amplified and single-molecule next-generation sequencing
    Article Snippet: We constructed sequencing libraries starting from 500 ng of total RNA. .. Purified RNA was dephosphorylated by adding 2 μL of 10× phosphatase buffer, 5 units of Antarctic phosphatase (NEB), and 40 units of RNaseOUT (Invitrogen) and incubating for 30 min at 37°C followed by 5 min at 65°C.

    Article Title: Improving the performance of true single molecule sequencing for ancient DNA
    Article Snippet: Paragraph title: tSMS sequencing ... For the phosphatase reactions, 0.5 μl of the DNA extracts were mixed with 8 μl of nuclease-free water, 1 μl of NEB Antarctic Phosphatase 10X Reaction buffer and 2.5 units of NEB Antarctic Phosphatase.

    Sonication:

    Article Title: BRCA1 Inhibits Membrane Estrogen and Growth Factor Receptor Signaling to Cell Proliferation in Breast Cancer
    Article Snippet: Two days earlier, a second set of MCF-7 cells had been transfected with pcDNA3 (control) or wt BRCA1 and then recovered and synchronized. .. The cells were washed twice with Dulbecco's minimal essential medium, scraped, and then sonicated in phosphatase activity buffer (New England Biolabs). .. After centrifugation, the supernatants (100 μl) from the cells subjected to each treatment were added in separate tubes to equal aliquots of 32 P-labeled ERK.

    Article Title:
    Article Snippet: Cells were lysed by sonication with two pulses of 5 s each, and the samples were subsequently cleared by ultracentrifugation at 13,000 × g if being used for Western blot analysis or 140,000 × g if being used for proteomic analysis. .. After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C.

    Article Title: RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases
    Article Snippet: Samples were solubilized for 1 min via sonication with a Covaris S220 instrument (Covaris, UK) using following parameters: Peak Incident Power (W): 140; Duty Factor: 5%; Cycles per Burst: 200. .. For dephosphorylation, 1× antarctic phosphatase reaction buffer (NEB, Germany) with 1 U/µL of antarctic phosphatase and 1 U/µL of RNase OUT (Invitrogen) were added and the suspension was incubated at 37°C for 30 min and 800 rpm.

    Injection:

    Article Title: Borrelia burgdorferi bb0426 encodes a 2?-deoxyribosyltransferase that plays a central role in purine salvage
    Article Snippet: A 50 μl aliquot of the DNA preparation was denatured (97°C for 2 min) and then immediately placed on ice. .. Ten microliters of 0.3M NaOAc pH 5.3, 5 μl 20 mM ZnSO4 and 10 μl P1 Nuclease (Sigma-Aldrich) were added to each sample and incubated at 37°C for 2 h. Next, 10 μl of 10× Antarctic phosphatase buffer and 2 μl (10 units) of Antarctic phosphatase (NEB, Ipswich, MA) were added and incubated at 37°C for 20 h. The reaction mixture was analysed by HPLC under the following conditions: 75 μl aliquot of sample was injected onto a Supelcosil LC-18-S 4.6 mm × 150 mm, 5 μm analytical column (Supelco) equilibrated with 50 mM potassium phosphate, pH 4.5, containing 8% methanol. .. Furthermore, spectral data were collected for each peak using a diode array detector scanning from 200 to 600 nm.

    Recombinant:

    Article Title: Cyanobacterial circadian clockwork: roles of KaiA, KaiB and the kaiBC promoter in regulating KaiC
    Article Snippet: For phosphatase treatment of purified recombinant KaiC, His6 -tagged KaiC protein was purified as described elsewhere ( ). .. About 1 µg of purified KaiC::His6 or 30 µg of total protein from extracts was diluted in 1× phosphatase buffer (New England Biolabs, Beverly, MA).

    Article Title: TBK1 Provides Context-Selective Support of the Activated AKT/mTOR Pathway in Lung Cancer
    Article Snippet: Samples were then heated to ≥ 95C for ≥ 5 min in SDS sample buffer (+β-me) and then separated by SDS-PAGE, followed by immunoblotting. .. Recombinant human S6K (Abnova) was first incubated with lambda protein phosphatase in phosphatase buffer (NEB) at a ratio of 3 units of phosphatase per 10ng of protein for 60 min at 30C. .. Following phosphatase treatment, 100ng of S6K was incubated with or without 50ng recombinant active TBK1 (EMD Millipore) in kinase buffer containing phosphatase inhibitors (25 mM Tris-HCl pH 7.4, 10mM MgCl2 , 0.1mM Na3 VO4 , 2mM DTT, 5mM β-glycerosphosphate) in the presence or absence of 200µM ATP for 30 min at 30C (Total reaction volume = 50µL).

    Article Title: Regulation of the filament structure and assembly of Acanthamoeba myosin II by phosphorylation of serines in the heavy-chain nonhelical tailpiece
    Article Snippet: Recombinant myosin (0.6 mg/mL) was dialyzed against 10 mM imidazole (pH 7.0) and 25 mM KCl overnight. .. An 40-µL aliquot was mixed with 5 µL of 10× concentrated phosphatase buffer (New England BioLabs) and 5 µL of 10× concentrated MnCl2 solution (New England BioLabs) and was incubated with 1 µL of lambda protein phosphatase (New England BioLabs) for 10 min at room temperature.

    Radioactivity:

    Article Title: Borrelia burgdorferi bb0426 encodes a 2?-deoxyribosyltransferase that plays a central role in purine salvage
    Article Snippet: Ten microliters of 0.3M NaOAc pH 5.3, 5 μl 20 mM ZnSO4 and 10 μl P1 Nuclease (Sigma-Aldrich) were added to each sample and incubated at 37°C for 2 h. Next, 10 μl of 10× Antarctic phosphatase buffer and 2 μl (10 units) of Antarctic phosphatase (NEB, Ipswich, MA) were added and incubated at 37°C for 20 h. The reaction mixture was analysed by HPLC under the following conditions: 75 μl aliquot of sample was injected onto a Supelcosil LC-18-S 4.6 mm × 150 mm, 5 μm analytical column (Supelco) equilibrated with 50 mM potassium phosphate, pH 4.5, containing 8% methanol. .. Peaks were identified by matching retention time with that of an authentic deoxynucleoside standard (dAdo, dC, dI, dGuo or dT), and further confirmation was made by matching corresponding spectral scans.

    RNA Sequencing Assay:

    Article Title: Comparison of CAGE and RNA-seq transcriptome profiling using clonally amplified and single-molecule next-generation sequencing
    Article Snippet: Paragraph title: RNA-seq ... Purified RNA was dephosphorylated by adding 2 μL of 10× phosphatase buffer, 5 units of Antarctic phosphatase (NEB), and 40 units of RNaseOUT (Invitrogen) and incubating for 30 min at 37°C followed by 5 min at 65°C.

    Magnetic Beads:

    Article Title: A second Wpl1 anti‐cohesion pathway requires dephosphorylation of fission yeast kleisin Rad21 by PP4
    Article Snippet: Lambda phosphatase treatment of Rad21 was performed on Rad21‐9PK immunoprecipitated from total cell extracts. .. Rad21‐9PK bound to magnetic beads was washed twice in phosphatase buffer (50 mM HEPES; 100 mM NaCl; 2 mM DTT; 0.01% Brij 35 pH 7.5) and beads dispensed into three 50‐μl aliquots (without phosphatase, 400 units phosphatase (New England Biolabs), 400 units phosphatase and 50 mM Na‐vanadate and 10 mM β‐glycerophosphate). .. MnCl2 was added to 1 mM and reactions were carried out for 40 min at 30°C, stopped by addition of Laemmli buffer and heating for 10 min at 95°C.

    Article Title: RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases
    Article Snippet: Immunoprecipitation was performed on a rotating wheel overnight at 4°C with rabbit IgG-conjugated Protein G magnetic beads (Invitrogen, Germany). .. For dephosphorylation, 1× antarctic phosphatase reaction buffer (NEB, Germany) with 1 U/µL of antarctic phosphatase and 1 U/µL of RNase OUT (Invitrogen) were added and the suspension was incubated at 37°C for 30 min and 800 rpm.

    Mutagenesis:

    Article Title: Parallel Regulation of von Hippel-Lindau Disease by pVHL-Mediated Degradation of B-Myb and Hypoxia-Inducible Factor α
    Article Snippet: 786-O cells (2.6 × 106 ) expressing 3×HA–B-Myb (wild type [WT]or Y15A mutant) or control cells were cultured in either one 10-cm dish (confluent condition) or two 15-cm dishes (sparse condition) for 2 days with one change of culture medium. .. Phosphatase treatment was performed as follows: 3×HA–B-Myb was immunoprecipitated and washed with phosphatase buffer supplied by New England BioLabs (NEB; Tokyo, Japan).

    Isolation:

    Article Title: Comparison of CAGE and RNA-seq transcriptome profiling using clonally amplified and single-molecule next-generation sequencing
    Article Snippet: Purified RNA was dephosphorylated by adding 2 μL of 10× phosphatase buffer, 5 units of Antarctic phosphatase (NEB), and 40 units of RNaseOUT (Invitrogen) and incubating for 30 min at 37°C followed by 5 min at 65°C. .. Purified RNA was dephosphorylated by adding 2 μL of 10× phosphatase buffer, 5 units of Antarctic phosphatase (NEB), and 40 units of RNaseOUT (Invitrogen) and incubating for 30 min at 37°C followed by 5 min at 65°C.

    Protein Kinase Assay:

    Article Title: Calcium-dependent protein kinases play an essential role in a plant defence response
    Article Snippet: Paragraph title: Immunocomplex protein kinase assay ... For in vitro interconversion experiments, reactions with NtCDPK2-myc immunoprecipitates/beads were washed once with phosphatase buffer (NEB) and resuspended in 100 µl of the same buffer containing 2 U of non-specific λ phosphatase (NEB) in the absence or presence (control) of 50 mM NaF and 10 mM Na3 VO4 .

    Labeling:

    Article Title: BRCA1 Inhibits Membrane Estrogen and Growth Factor Receptor Signaling to Cell Proliferation in Breast Cancer
    Article Snippet: This complex was extensively washed to remove unincorporated 32 P. After protein determination, equal amounts of labeled ERK were used as a substrate for determining phosphatase activity under various treatment conditions. .. The cells were washed twice with Dulbecco's minimal essential medium, scraped, and then sonicated in phosphatase activity buffer (New England Biolabs).

    Article Title:
    Article Snippet: Lysates were then exchanged into 100 mm HEPES (pH 7.0) 3× using 3 K cut-off buffer exchange filters (Amicon, Billerica, MA) to remove phosphatase inhibitors prior to λ phosphatase treatment and small-molecule primary amines to permit complete dimethyl labeling of primary amines on proteins. .. After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C.

    Article Title: RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases
    Article Snippet: For dephosphorylation, 1× antarctic phosphatase reaction buffer (NEB, Germany) with 1 U/µL of antarctic phosphatase and 1 U/µL of RNase OUT (Invitrogen) were added and the suspension was incubated at 37°C for 30 min and 800 rpm. .. For dephosphorylation, 1× antarctic phosphatase reaction buffer (NEB, Germany) with 1 U/µL of antarctic phosphatase and 1 U/µL of RNase OUT (Invitrogen) were added and the suspension was incubated at 37°C for 30 min and 800 rpm.

    Purification:

    Article Title: Cyanobacterial circadian clockwork: roles of KaiA, KaiB and the kaiBC promoter in regulating KaiC
    Article Snippet: For phosphatase treatment of purified recombinant KaiC, His6 -tagged KaiC protein was purified as described elsewhere ( ). .. About 1 µg of purified KaiC::His6 or 30 µg of total protein from extracts was diluted in 1× phosphatase buffer (New England Biolabs, Beverly, MA). .. For the samples that included phosphatase, 1000 U of lambda protein phosphatase (λPPase; New England Biolabs) were added, and the reaction was incubated at 30°C for 1 h in the presence or absence of 40 mM sodium vanadate.

    Article Title: Comparison of CAGE and RNA-seq transcriptome profiling using clonally amplified and single-molecule next-generation sequencing
    Article Snippet: Fragment RNA was purified with the RNeasy MinElute kit (Qiagen) following the instructions of the manufacturer except 675 μL of 100% ethanol was used in step 2. .. Purified RNA was dephosphorylated by adding 2 μL of 10× phosphatase buffer, 5 units of Antarctic phosphatase (NEB), and 40 units of RNaseOUT (Invitrogen) and incubating for 30 min at 37°C followed by 5 min at 65°C. .. After incubation, the sample was set on ice and 5 μL of 10× PNK buffer, 20 units of T4 polynucleotide kinase (NEB), 5 μL of 10 mM ATP (Epicentre), 40 units of RNaseOUT, and 17 μL of water were added, and incubated at 37°C for 60 min. Phosphorated RNA was purified with the RNeasy MinElute kit (Qiagen) as described before.

    Article Title: Feeder-Free Derivation of Human Induced Pluripotent Stem Cells with Messenger RNA
    Article Snippet: Reactions were incubated 4–6 hours at 37°C and treated with 2 uL TURBO DNase for a further 15 minutes at 37°C before being purified on MEGAclear (Ambion) spin columns, the RNA products being eluted in a volume of 100 uL. .. To remove immunogenic 5′ triphosphate moieties from uncapped transcripts, 10 uL of Antarctic Phosphatase reaction buffer and 3 uL of Antarctic Phosphatase (NEB) was added to each prep.

    Article Title: mRNA Transfection of Mouse and Human Neural Stem Cell Cultures
    Article Snippet: Reactions were incubated 3-4 hours at 37°C then treated with 1 µl TURBO DNase (Life Technologies) for a further 15-minutes at 37°C before purification on RNeasy MiniSpin columns (Qiagen). .. To remove 5′ triphosphate moieties from uncapped transcripts, 10 µl of Antarctic Phosphatase reaction buffer and 5 µl (25U) of Antarctic Phosphatase (New England BioLabs) was added to each RNA product and incubated for 30-minutes at 37°C.

    Article Title: Phosphatase 1 Nuclear Targeting Subunit Is an Essential Regulator of M-phase Entry, Maintenance, and Exit
    Article Snippet: Purified histone H3 peptide was phosphorylated with Aurora A kinase (a gift from Dr. M. Y. Tsai) in kinase buffer (20 m m HEPES, pH 7.5, 2 m m DTT, 10 m m MgCl2 , 0.1 m m EGTA, 100 μ m ATP) for 30 min at 30 °C. .. For the PP1 phosphatase assay, prephosphorylated histone H3 peptide was incubated with PP1 (New England Biolabs) in phosphatase buffer (New England Biolabs) with and without Pnuts protein at room temperature for the time indicated.

    Article Title: High-Resolution Optical Tweezers Combined With Single-Molecule Confocal Microscopy
    Article Snippet: Incubate the LH reaction mix at 75°C for 1 h. Add the following to the ~35 μL of digested LH: 4 μL 10 × Antarctic phosphatase buffer (NEB), 1 μL Antarctic phosphatase (5 units; NEB). .. Incubate the LH reaction mix at 75°C for 1 h. Add the following to the ~35 μL of digested LH: 4 μL 10 × Antarctic phosphatase buffer (NEB), 1 μL Antarctic phosphatase (5 units; NEB).

    Polymerase Chain Reaction:

    Article Title: High-Resolution Optical Tweezers Combined With Single-Molecule Confocal Microscopy
    Article Snippet: Incubate the LH reaction mix at 75°C for 1 h. Add the following to the ~35 μL of digested LH: 4 μL 10 × Antarctic phosphatase buffer (NEB), 1 μL Antarctic phosphatase (5 units; NEB). .. Incubate the LH reaction mix at 75°C for 1 h. Add the following to the ~35 μL of digested LH: 4 μL 10 × Antarctic phosphatase buffer (NEB), 1 μL Antarctic phosphatase (5 units; NEB).

    Immunoprecipitation:

    Article Title: Orm family proteins mediate sphingolipid homeostasis
    Article Snippet: Supernatants were then added to anti-Flag agarose (25µl bed volume) and immunoprecipitated for 2.5 hr at 4°C. .. The washed resin was then resuspended in 54µl phosphatase buffer + 6µl calf intestine phosphatase (New England Biolabs) and incubated for 1 hr at 37°C.

    Article Title: Phosphorylation regulates human polη stability and damage bypass throughout the cell cycle
    Article Snippet: A total of 120 μg protein extracts were incubated in phosphatase buffer (New England Biolabs) with or without 1600U of λ phosphatase (New England Biolabs) for 30 min at 30°C. .. After incubation the reaction was diluted with rehydration buffer up to a volume of 200 μl and processed as previously described for 2D-PAGE.

    Article Title: BRCA1 Inhibits Membrane Estrogen and Growth Factor Receptor Signaling to Cell Proliferation in Breast Cancer
    Article Snippet: The cells were lysed, and the lysate was subjected to immunoprecipitation with agarose bead-conjugated polyclonal antibody against ERK2 (Santa Cruz). .. The cells were washed twice with Dulbecco's minimal essential medium, scraped, and then sonicated in phosphatase activity buffer (New England Biolabs).

    Article Title: Parallel Regulation of von Hippel-Lindau Disease by pVHL-Mediated Degradation of B-Myb and Hypoxia-Inducible Factor α
    Article Snippet: For tyrosine kinase inhibition, the following inhibitors were added to the culture medium for 1 day before harvesting the cells: 0.1 μM epidermal growth factor receptor (EGFR) inhibitor PD153035, 5 μM ErbB2 inhibitor AG825, 0.1 μM insulin-like growth factor receptor (IGFR) inhibitor picropodophyllin, 1 μM mesenchymal epithelial transition factor (MET) inhibitor SU11274, and 5 μM vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) inhibitor SU4312. .. Phosphatase treatment was performed as follows: 3×HA–B-Myb was immunoprecipitated and washed with phosphatase buffer supplied by New England BioLabs (NEB; Tokyo, Japan). .. Immunoprecipitates were incubated with 10 U of Antarctic phosphatase (M0289; NEB) for 30 min at 37°C.

    Article Title: A second Wpl1 anti‐cohesion pathway requires dephosphorylation of fission yeast kleisin Rad21 by PP4
    Article Snippet: Lambda phosphatase treatment of Rad21 was performed on Rad21‐9PK immunoprecipitated from total cell extracts. .. Rad21‐9PK bound to magnetic beads was washed twice in phosphatase buffer (50 mM HEPES; 100 mM NaCl; 2 mM DTT; 0.01% Brij 35 pH 7.5) and beads dispensed into three 50‐μl aliquots (without phosphatase, 400 units phosphatase (New England Biolabs), 400 units phosphatase and 50 mM Na‐vanadate and 10 mM β‐glycerophosphate).

    Article Title: RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases
    Article Snippet: Immunoprecipitated and crosslinked RNA was partially digested with 50 U of RNase T1 per mL for 20 min at 25°C and 400 rpm. .. For dephosphorylation, 1× antarctic phosphatase reaction buffer (NEB, Germany) with 1 U/µL of antarctic phosphatase and 1 U/µL of RNase OUT (Invitrogen) were added and the suspension was incubated at 37°C for 30 min and 800 rpm.

    De-Phosphorylation Assay:

    Article Title: Regulation of the filament structure and assembly of Acanthamoeba myosin II by phosphorylation of serines in the heavy-chain nonhelical tailpiece
    Article Snippet: Paragraph title: Phosphorylation of Filamentous WT and Dephosphorylation of Filamentous pWT. ... An 40-µL aliquot was mixed with 5 µL of 10× concentrated phosphatase buffer (New England BioLabs) and 5 µL of 10× concentrated MnCl2 solution (New England BioLabs) and was incubated with 1 µL of lambda protein phosphatase (New England BioLabs) for 10 min at room temperature.

    Article Title: RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases
    Article Snippet: Beads were washed twice in T1 buffer and phosphatase reaction buffer (50 mM Tris-HCl pH 7.0, 1 mM MgCl2 , 0.1 mM ZnCl2 ). .. For dephosphorylation, 1× antarctic phosphatase reaction buffer (NEB, Germany) with 1 U/µL of antarctic phosphatase and 1 U/µL of RNase OUT (Invitrogen) were added and the suspension was incubated at 37°C for 30 min and 800 rpm. .. Beads were washed once in phosphatase wash buffer (50 mM Tris-HCl pH 7.5, 20 mM EGTA, 0.5% NP-40) and twice in polynucleotide kinase (PNK) buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM MgCl2 ).

    Lysis:

    Article Title: Orm family proteins mediate sphingolipid homeostasis
    Article Snippet: 200µl acid-washed glass beads and 150µl SDS lysis buffer (50mM Tris-Cl, pH 7.8, 1mM EDTA, 1% SDS, 2M Urea) supplemented with protease inhibitors were added to the dried pellets, which were then lysed by bead-beating (3× 60s at 4°C). .. The washed resin was then resuspended in 54µl phosphatase buffer + 6µl calf intestine phosphatase (New England Biolabs) and incubated for 1 hr at 37°C.

    Article Title: The Hem protein mediates neuronal migration by inhibiting WAVE degradation and functions opposite of Abelson tyrosine kinase
    Article Snippet: Twenty embryos were collected, dechorionated and homogenized in 37.5μL of the Lysis buffer (see above). .. After centrifugation, supernatant (~37.5μL) was collected and incubated with Lambda protein phosphatase (Lambda PP, 100U) in NEB phosphatase buffer (5μL of 10X) and MnCl2 (5μL of 10 mM solution) in a total final volume of 50μL for 30 min at 30°C.

    Liquid Chromatography:

    Article Title: Borrelia burgdorferi bb0426 encodes a 2?-deoxyribosyltransferase that plays a central role in purine salvage
    Article Snippet: A 50 μl aliquot of the DNA preparation was denatured (97°C for 2 min) and then immediately placed on ice. .. Ten microliters of 0.3M NaOAc pH 5.3, 5 μl 20 mM ZnSO4 and 10 μl P1 Nuclease (Sigma-Aldrich) were added to each sample and incubated at 37°C for 2 h. Next, 10 μl of 10× Antarctic phosphatase buffer and 2 μl (10 units) of Antarctic phosphatase (NEB, Ipswich, MA) were added and incubated at 37°C for 20 h. The reaction mixture was analysed by HPLC under the following conditions: 75 μl aliquot of sample was injected onto a Supelcosil LC-18-S 4.6 mm × 150 mm, 5 μm analytical column (Supelco) equilibrated with 50 mM potassium phosphate, pH 4.5, containing 8% methanol. .. Furthermore, spectral data were collected for each peak using a diode array detector scanning from 200 to 600 nm.

    Electrophoresis:

    Article Title: mRNA Transfection of Mouse and Human Neural Stem Cell Cultures
    Article Snippet: To remove 5′ triphosphate moieties from uncapped transcripts, 10 µl of Antarctic Phosphatase reaction buffer and 5 µl (25U) of Antarctic Phosphatase (New England BioLabs) was added to each RNA product and incubated for 30-minutes at 37°C. .. Synthesized RNA products were then repurified using RNeasy Mini Columns (Qiagen) and quantitated by spectrophotometry and denaturing gel electrophoresis.

    In Vitro:

    Article Title: Calcium-dependent protein kinases play an essential role in a plant defence response
    Article Snippet: Subsequently, samples were aliquoted (5 µl of beads each), washed with kinase buffer and resuspended in 5 µl of kinase buffer (40 mM HEPES pH 7.4, 10 mM MgCl2 , 2 mM DTT, 0.1 mM EGTA). .. For in vitro interconversion experiments, reactions with NtCDPK2-myc immunoprecipitates/beads were washed once with phosphatase buffer (NEB) and resuspended in 100 µl of the same buffer containing 2 U of non-specific λ phosphatase (NEB) in the absence or presence (control) of 50 mM NaF and 10 mM Na3 VO4 . .. After 10 min at 37°C the reaction was stopped by addition of NaF and Na3 VO4 , and the beads were washed twice to separate from residual (although inhibited) phosphatase and resuspended in 5 µl of kinase buffer.

    Article Title: TBK1 Provides Context-Selective Support of the Activated AKT/mTOR Pathway in Lung Cancer
    Article Snippet: Paragraph title: In Vitro Kinase Assay ... Recombinant human S6K (Abnova) was first incubated with lambda protein phosphatase in phosphatase buffer (NEB) at a ratio of 3 units of phosphatase per 10ng of protein for 60 min at 30C.

    Concentration Assay:

    Article Title: Calcium-dependent protein kinases play an essential role in a plant defence response
    Article Snippet: For in vitro interconversion experiments, reactions with NtCDPK2-myc immunoprecipitates/beads were washed once with phosphatase buffer (NEB) and resuspended in 100 µl of the same buffer containing 2 U of non-specific λ phosphatase (NEB) in the absence or presence (control) of 50 mM NaF and 10 mM Na3 VO4 . .. For in vitro interconversion experiments, reactions with NtCDPK2-myc immunoprecipitates/beads were washed once with phosphatase buffer (NEB) and resuspended in 100 µl of the same buffer containing 2 U of non-specific λ phosphatase (NEB) in the absence or presence (control) of 50 mM NaF and 10 mM Na3 VO4 .

    Article Title: Feeder-Free Derivation of Human Induced Pluripotent Stem Cells with Messenger RNA
    Article Snippet: To remove immunogenic 5′ triphosphate moieties from uncapped transcripts, 10 uL of Antarctic Phosphatase reaction buffer and 3 uL of Antarctic Phosphatase (NEB) was added to each prep. .. Phosphatase reactions were incubated for 30 minutes at 37°C and the IVT products were repurified.

    Article Title:
    Article Snippet: After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C. .. After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C.

    Article Title: RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases
    Article Snippet: For dephosphorylation, 1× antarctic phosphatase reaction buffer (NEB, Germany) with 1 U/µL of antarctic phosphatase and 1 U/µL of RNase OUT (Invitrogen) were added and the suspension was incubated at 37°C for 30 min and 800 rpm. .. For dephosphorylation, 1× antarctic phosphatase reaction buffer (NEB, Germany) with 1 U/µL of antarctic phosphatase and 1 U/µL of RNase OUT (Invitrogen) were added and the suspension was incubated at 37°C for 30 min and 800 rpm.

    Staining:

    Article Title: Cyanobacterial circadian clockwork: roles of KaiA, KaiB and the kaiBC promoter in regulating KaiC
    Article Snippet: About 1 µg of purified KaiC::His6 or 30 µg of total protein from extracts was diluted in 1× phosphatase buffer (New England Biolabs, Beverly, MA). .. For the samples that included phosphatase, 1000 U of lambda protein phosphatase (λPPase; New England Biolabs) were added, and the reaction was incubated at 30°C for 1 h in the presence or absence of 40 mM sodium vanadate.

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    New England Biolabs 10x antarctic phosphatase reaction buffer
    10x Antarctic Phosphatase Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/10x antarctic phosphatase reaction buffer/product/New England Biolabs
    Average 99 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    10x antarctic phosphatase reaction buffer - by Bioz Stars, 2019-10
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs 10x antarctic phosphatase buffer
    10x Antarctic Phosphatase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/10x antarctic phosphatase buffer/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    10x antarctic phosphatase buffer - by Bioz Stars, 2019-10
    99/100 stars
      Buy from Supplier

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