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phosphatase buffer  (New England Biolabs)


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    New England Biolabs phosphatase buffer
    (A) Western blot analysis of cleared bacterial lysates of GST-PFKFB2 and mutants. Top: Total protein staining of cleared bacterial lysates over-expressing GST-PFKFB2 and mutants. Bottom: Western blots against GST and phosphorylated-PFKFB2 (Ser483) for GST-PFKFB2 and mutants. “Double mut” indicates protein containing both K174G and H259A. (B) Western blot analysis of GST-PFKFB2 expression, purification, and in vitro phosphorylation. Flow thru indicates proteins unbound to GSH beads. Column are proteins bound to the GSH beads. Column + Thrombin are proteins remaining bound to GSH beads after thrombin cleavage. Purified PFK2 lanes are proteins eluted from the GSH beads with thrombin cleavage. Purified PFK2 + ATP are proteins incubated with ATP alone. Purified PFK2 + ATP + PKA are proteins phosphorylated by PKA. (C) Western blot analysis of GST-PFKFB2 and mutant proteins showing they are phosphorylated (Ser483) upon extraction from bacteria ( left lanes ), lose phosphorylation after purification ( middle lanes ), but are re-phosphorylated upon treatment with PKA and ATP ( right lanes ). (D) Representative western blot against phosphorylated-PFKFB2 (Ser483) of bacterial samples (WT PFKFB2) treated/not treated with <t>λ-phosphatase</t> for indicated times before lysis. Equal amounts of purified PFKFB2 were treated and loaded into each lane. Quantitation of bacterial samples treated with λ-phosphatase. Data are shown as mean±SD. The original, unedited blots are included in .
    Phosphatase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2917 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/lambda+protein+phosphatase+buffer/Lambda+Protein+Phosphatase/pmc12097583-49-16-21
    Average 99 stars, based on 2917 article reviews
    phosphatase buffer - by Bioz Stars, 2026-10
    99/100 stars

    Images

    1) Product Images from "Mechanistic studies of PFKFB2 reveal a novel inhibitor of its kinase activity"

    Article Title: Mechanistic studies of PFKFB2 reveal a novel inhibitor of its kinase activity

    Journal: PLOS One

    doi: 10.1371/journal.pone.0317167

    (A) Western blot analysis of cleared bacterial lysates of GST-PFKFB2 and mutants. Top: Total protein staining of cleared bacterial lysates over-expressing GST-PFKFB2 and mutants. Bottom: Western blots against GST and phosphorylated-PFKFB2 (Ser483) for GST-PFKFB2 and mutants. “Double mut” indicates protein containing both K174G and H259A. (B) Western blot analysis of GST-PFKFB2 expression, purification, and in vitro phosphorylation. Flow thru indicates proteins unbound to GSH beads. Column are proteins bound to the GSH beads. Column + Thrombin are proteins remaining bound to GSH beads after thrombin cleavage. Purified PFK2 lanes are proteins eluted from the GSH beads with thrombin cleavage. Purified PFK2 + ATP are proteins incubated with ATP alone. Purified PFK2 + ATP + PKA are proteins phosphorylated by PKA. (C) Western blot analysis of GST-PFKFB2 and mutant proteins showing they are phosphorylated (Ser483) upon extraction from bacteria ( left lanes ), lose phosphorylation after purification ( middle lanes ), but are re-phosphorylated upon treatment with PKA and ATP ( right lanes ). (D) Representative western blot against phosphorylated-PFKFB2 (Ser483) of bacterial samples (WT PFKFB2) treated/not treated with λ-phosphatase for indicated times before lysis. Equal amounts of purified PFKFB2 were treated and loaded into each lane. Quantitation of bacterial samples treated with λ-phosphatase. Data are shown as mean±SD. The original, unedited blots are included in .
    Figure Legend Snippet: (A) Western blot analysis of cleared bacterial lysates of GST-PFKFB2 and mutants. Top: Total protein staining of cleared bacterial lysates over-expressing GST-PFKFB2 and mutants. Bottom: Western blots against GST and phosphorylated-PFKFB2 (Ser483) for GST-PFKFB2 and mutants. “Double mut” indicates protein containing both K174G and H259A. (B) Western blot analysis of GST-PFKFB2 expression, purification, and in vitro phosphorylation. Flow thru indicates proteins unbound to GSH beads. Column are proteins bound to the GSH beads. Column + Thrombin are proteins remaining bound to GSH beads after thrombin cleavage. Purified PFK2 lanes are proteins eluted from the GSH beads with thrombin cleavage. Purified PFK2 + ATP are proteins incubated with ATP alone. Purified PFK2 + ATP + PKA are proteins phosphorylated by PKA. (C) Western blot analysis of GST-PFKFB2 and mutant proteins showing they are phosphorylated (Ser483) upon extraction from bacteria ( left lanes ), lose phosphorylation after purification ( middle lanes ), but are re-phosphorylated upon treatment with PKA and ATP ( right lanes ). (D) Representative western blot against phosphorylated-PFKFB2 (Ser483) of bacterial samples (WT PFKFB2) treated/not treated with λ-phosphatase for indicated times before lysis. Equal amounts of purified PFKFB2 were treated and loaded into each lane. Quantitation of bacterial samples treated with λ-phosphatase. Data are shown as mean±SD. The original, unedited blots are included in .

    Techniques Used: Western Blot, Staining, Expressing, Purification, In Vitro, Phospho-proteomics, Incubation, Mutagenesis, Extraction, Bacteria, Lysis, Quantitation Assay

    Related Articles

    Construct:

    Article Title: Exploration of targeted electrophilic kinase probes identifies a covalent ULK1 degrader
    Article Snippet: .. ULK1-encoding constructs were co-transformed with a plasmid encoding lambda protein phosphatase in E. coli Lemo21 (DE3) strain (New England Biolabs, #C2528J) and grown at 30 °C with selection antibiotics (50 μg/mL kanamycin, 100 μg/mL ampicillin, 30 μg/mL chloramphenicol) in presence of 50 μM rhamnose to repress protein expression in absence of IPTG. ..

    Plasmid Preparation:

    Article Title: Exploration of targeted electrophilic kinase probes identifies a covalent ULK1 degrader
    Article Snippet: .. ULK1-encoding constructs were co-transformed with a plasmid encoding lambda protein phosphatase in E. coli Lemo21 (DE3) strain (New England Biolabs, #C2528J) and grown at 30 °C with selection antibiotics (50 μg/mL kanamycin, 100 μg/mL ampicillin, 30 μg/mL chloramphenicol) in presence of 50 μM rhamnose to repress protein expression in absence of IPTG. ..

    Selection:

    Article Title: Exploration of targeted electrophilic kinase probes identifies a covalent ULK1 degrader
    Article Snippet: .. ULK1-encoding constructs were co-transformed with a plasmid encoding lambda protein phosphatase in E. coli Lemo21 (DE3) strain (New England Biolabs, #C2528J) and grown at 30 °C with selection antibiotics (50 μg/mL kanamycin, 100 μg/mL ampicillin, 30 μg/mL chloramphenicol) in presence of 50 μM rhamnose to repress protein expression in absence of IPTG. ..

    Expressing:

    Article Title: Exploration of targeted electrophilic kinase probes identifies a covalent ULK1 degrader
    Article Snippet: .. ULK1-encoding constructs were co-transformed with a plasmid encoding lambda protein phosphatase in E. coli Lemo21 (DE3) strain (New England Biolabs, #C2528J) and grown at 30 °C with selection antibiotics (50 μg/mL kanamycin, 100 μg/mL ampicillin, 30 μg/mL chloramphenicol) in presence of 50 μM rhamnose to repress protein expression in absence of IPTG. ..

    Affinity Chromatography:

    Article Title: GluDs are ionotropic dopamine receptors tuned by G-proteins
    Article Snippet: .. Following reverse Ni affinity chromatography to remove uncleaved, His-tagged protein and 3C protease, the flowthrough solution, supplemented with 1 mM manganese chloride, was collected and treated with CIP (NEB, M0525S), Antarctic phosphatase (NEB, M0289S), and λ phosphatase (NEB, P0753S) for 1 hour at 4 °C to dephosphorylate the protein. .. After adjusting the NaCl concentration to 50 mM by adding dilution buffer composed of 20 mM HEPES pH 7.4, 1 mM MgCl 2 , 10 μM GDP, 100 μM TCEP, and 0.05% DDM, the protein was loaded onto a Capto HiRes Q 10/100 (Cytiva) anion exchange column equilibrated in buffer A composed of 20 mM HEPES pH 7.4, 50 mM NaCl, 1 mM MgCl 2 , 10 μM GDP, 100 μM TCEP, and 0.05% DDM.

    Recombinant:

    Article Title: A tandem recruitment site in the pseudokinase scaffold PEAK3 is subject to phosphorylation-dependent regulation and cancer-associated mutations
    Article Snippet: Goat anti-rabbit IgG (H+L) secondary antibody conjugated to Alexa Fluor 555 (A-21428) was from ThermoFisher Scientific. .. The following reagents/inhibitors were used in this study: lambda protein phosphatase (New England Biolabs, P0753S), animal-free recombinant human EGF (PeproTech, AF-100–15), insulin from bovine pancreas (Merck, I-1882), CaMKII inhibitor RA306 (custom synthesized by Reagency, RGNCY-0117, 1 μM final), PKA Inhibitor 14 to 22 Amide, Cell-Permeable, Myristoylated (Calbiochem, 476,485, 10 μM final), DMSO (Sigma, d8418, 0.1% final), and MedChemExpress-sourced Akt inhibitor MK-2206 dihydrochloride (HY-10358, 100 nM final) and trametinib (HY-10999, 10 nM final). .. PKC inhibitor Go6850 (S7208, 10 μM final) and Go6976 (S7119, 5 μM final), Pan PI3K inhibitor BKM120 (also known as Buparlisib/NVP-BKM120, S2247, 500 nM final), ROCK inhibitor (Y-27632, 10 μM final), and PKD inhibitor CRT0066101 dihydrochloride (S8366, 10 μM final) were purchased from Selleck Chemicals.

    Synthesized:

    Article Title: A tandem recruitment site in the pseudokinase scaffold PEAK3 is subject to phosphorylation-dependent regulation and cancer-associated mutations
    Article Snippet: Goat anti-rabbit IgG (H+L) secondary antibody conjugated to Alexa Fluor 555 (A-21428) was from ThermoFisher Scientific. .. The following reagents/inhibitors were used in this study: lambda protein phosphatase (New England Biolabs, P0753S), animal-free recombinant human EGF (PeproTech, AF-100–15), insulin from bovine pancreas (Merck, I-1882), CaMKII inhibitor RA306 (custom synthesized by Reagency, RGNCY-0117, 1 μM final), PKA Inhibitor 14 to 22 Amide, Cell-Permeable, Myristoylated (Calbiochem, 476,485, 10 μM final), DMSO (Sigma, d8418, 0.1% final), and MedChemExpress-sourced Akt inhibitor MK-2206 dihydrochloride (HY-10358, 100 nM final) and trametinib (HY-10999, 10 nM final). .. PKC inhibitor Go6850 (S7208, 10 μM final) and Go6976 (S7119, 5 μM final), Pan PI3K inhibitor BKM120 (also known as Buparlisib/NVP-BKM120, S2247, 500 nM final), ROCK inhibitor (Y-27632, 10 μM final), and PKD inhibitor CRT0066101 dihydrochloride (S8366, 10 μM final) were purchased from Selleck Chemicals.

    De-Phosphorylation Assay:

    Article Title: Phosphorylation by CDK1 switches the function of the human SLX4 SAP domain from DNA binding to MUS81 binding
    Article Snippet: The protocol described in Ref [ ] was used to phosphorylate with CDK1-CycB1 (Lifetechnology) and purify the rSAP and rSAP4TA proteins. .. Dephosphorylation was achieved by incubating samples for 1 h at 30°C with 1 μl of lambda phosphatase (NEB) for 9 μM of SAP, 1X phosphatase buffer, and 1 mM MgCl 2 , in a final volume of 10 μl. ..



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    (A) Western blot analysis of cleared bacterial lysates of GST-PFKFB2 and mutants. Top: Total protein staining of cleared bacterial lysates over-expressing GST-PFKFB2 and mutants. Bottom: Western blots against GST and phosphorylated-PFKFB2 (Ser483) for GST-PFKFB2 and mutants. “Double mut” indicates protein containing both K174G and H259A. (B) Western blot analysis of GST-PFKFB2 expression, purification, and in vitro phosphorylation. Flow thru indicates proteins unbound to GSH beads. Column are proteins bound to the GSH beads. Column + Thrombin are proteins remaining bound to GSH beads after thrombin cleavage. Purified PFK2 lanes are proteins eluted from the GSH beads with thrombin cleavage. Purified PFK2 + ATP are proteins incubated with ATP alone. Purified PFK2 + ATP + PKA are proteins phosphorylated by PKA. (C) Western blot analysis of GST-PFKFB2 and mutant proteins showing they are phosphorylated (Ser483) upon extraction from bacteria ( left lanes ), lose phosphorylation after purification ( middle lanes ), but are re-phosphorylated upon treatment with PKA and ATP ( right lanes ). (D) Representative western blot against phosphorylated-PFKFB2 (Ser483) of bacterial samples (WT PFKFB2) treated/not treated with <t>λ-phosphatase</t> for indicated times before lysis. Equal amounts of purified PFKFB2 were treated and loaded into each lane. Quantitation of bacterial samples treated with λ-phosphatase. Data are shown as mean±SD. The original, unedited blots are included in .
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    (A) Western blot analysis of cleared bacterial lysates of GST-PFKFB2 and mutants. Top: Total protein staining of cleared bacterial lysates over-expressing GST-PFKFB2 and mutants. Bottom: Western blots against GST and phosphorylated-PFKFB2 (Ser483) for GST-PFKFB2 and mutants. “Double mut” indicates protein containing both K174G and H259A. (B) Western blot analysis of GST-PFKFB2 expression, purification, and in vitro phosphorylation. Flow thru indicates proteins unbound to GSH beads. Column are proteins bound to the GSH beads. Column + Thrombin are proteins remaining bound to GSH beads after thrombin cleavage. Purified PFK2 lanes are proteins eluted from the GSH beads with thrombin cleavage. Purified PFK2 + ATP are proteins incubated with ATP alone. Purified PFK2 + ATP + PKA are proteins phosphorylated by PKA. (C) Western blot analysis of GST-PFKFB2 and mutant proteins showing they are phosphorylated (Ser483) upon extraction from bacteria ( left lanes ), lose phosphorylation after purification ( middle lanes ), but are re-phosphorylated upon treatment with PKA and ATP ( right lanes ). (D) Representative western blot against phosphorylated-PFKFB2 (Ser483) of bacterial samples (WT PFKFB2) treated/not treated with <t>λ-phosphatase</t> for indicated times before lysis. Equal amounts of purified PFKFB2 were treated and loaded into each lane. Quantitation of bacterial samples treated with λ-phosphatase. Data are shown as mean±SD. The original, unedited blots are included in .
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    Image Search Results


    (A) Western blot analysis of cleared bacterial lysates of GST-PFKFB2 and mutants. Top: Total protein staining of cleared bacterial lysates over-expressing GST-PFKFB2 and mutants. Bottom: Western blots against GST and phosphorylated-PFKFB2 (Ser483) for GST-PFKFB2 and mutants. “Double mut” indicates protein containing both K174G and H259A. (B) Western blot analysis of GST-PFKFB2 expression, purification, and in vitro phosphorylation. Flow thru indicates proteins unbound to GSH beads. Column are proteins bound to the GSH beads. Column + Thrombin are proteins remaining bound to GSH beads after thrombin cleavage. Purified PFK2 lanes are proteins eluted from the GSH beads with thrombin cleavage. Purified PFK2 + ATP are proteins incubated with ATP alone. Purified PFK2 + ATP + PKA are proteins phosphorylated by PKA. (C) Western blot analysis of GST-PFKFB2 and mutant proteins showing they are phosphorylated (Ser483) upon extraction from bacteria ( left lanes ), lose phosphorylation after purification ( middle lanes ), but are re-phosphorylated upon treatment with PKA and ATP ( right lanes ). (D) Representative western blot against phosphorylated-PFKFB2 (Ser483) of bacterial samples (WT PFKFB2) treated/not treated with λ-phosphatase for indicated times before lysis. Equal amounts of purified PFKFB2 were treated and loaded into each lane. Quantitation of bacterial samples treated with λ-phosphatase. Data are shown as mean±SD. The original, unedited blots are included in .

    Journal: PLOS One

    Article Title: Mechanistic studies of PFKFB2 reveal a novel inhibitor of its kinase activity

    doi: 10.1371/journal.pone.0317167

    Figure Lengend Snippet: (A) Western blot analysis of cleared bacterial lysates of GST-PFKFB2 and mutants. Top: Total protein staining of cleared bacterial lysates over-expressing GST-PFKFB2 and mutants. Bottom: Western blots against GST and phosphorylated-PFKFB2 (Ser483) for GST-PFKFB2 and mutants. “Double mut” indicates protein containing both K174G and H259A. (B) Western blot analysis of GST-PFKFB2 expression, purification, and in vitro phosphorylation. Flow thru indicates proteins unbound to GSH beads. Column are proteins bound to the GSH beads. Column + Thrombin are proteins remaining bound to GSH beads after thrombin cleavage. Purified PFK2 lanes are proteins eluted from the GSH beads with thrombin cleavage. Purified PFK2 + ATP are proteins incubated with ATP alone. Purified PFK2 + ATP + PKA are proteins phosphorylated by PKA. (C) Western blot analysis of GST-PFKFB2 and mutant proteins showing they are phosphorylated (Ser483) upon extraction from bacteria ( left lanes ), lose phosphorylation after purification ( middle lanes ), but are re-phosphorylated upon treatment with PKA and ATP ( right lanes ). (D) Representative western blot against phosphorylated-PFKFB2 (Ser483) of bacterial samples (WT PFKFB2) treated/not treated with λ-phosphatase for indicated times before lysis. Equal amounts of purified PFKFB2 were treated and loaded into each lane. Quantitation of bacterial samples treated with λ-phosphatase. Data are shown as mean±SD. The original, unedited blots are included in .

    Article Snippet: Assay conditions were 100 µL sonicated sample, 10 µL MnCl 2 (10 mM NEB), 10 µL phosphatase buffer (10x PMP buffer, NEB) with or without 2 µL Lambda protein phosphatase (NEB, 400000 U/mL).

    Techniques: Western Blot, Staining, Expressing, Purification, In Vitro, Phospho-proteomics, Incubation, Mutagenesis, Extraction, Bacteria, Lysis, Quantitation Assay