phosphatase reaction  (New England Biolabs)


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    Name:
    Antarctic Phosphatase Reaction Buffer
    Description:
    Antarctic Phosphatase Reaction Buffer 6 0 ml
    Catalog Number:
    B0289S
    Price:
    24
    Category:
    Buffers
    Size:
    6 0 ml
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    New England Biolabs phosphatase reaction
    Antarctic Phosphatase Reaction Buffer
    Antarctic Phosphatase Reaction Buffer 6 0 ml
    https://www.bioz.com/result/phosphatase reaction/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phosphatase reaction - by Bioz Stars, 2021-09
    95/100 stars

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    other:

    Article Title: Phosphorylation of the LIR Domain of SCOC Modulates ATG8 Binding Affinity and Specificity
    Article Snippet: Lambda phosphatase was added into each sample with phosphatase reaction buffer and MnCl2 following manufacturer’s instructions (New England BioLabs).

    Article Title: Control of topoisomerase II activity and chemotherapeutic inhibition by TCA cycle metabolites
    Article Snippet: Next, 5μl of 10X reaction buffer was added to each sample: 10x Antarctic phosphatase (AP) reaction buffer (New England BioLabs) was added to two samples and 200 mM Tris-HCl (pH 7.9), 1 M NaCl, 200 mM MgCl2 was added to the remaining two samples for SVPD treatment.

    Article Title: A cytoplasmic protein kinase in Chlamydomonas couples engagement of ciliary receptors to rapid cellular responses
    Article Snippet: The 40 μl final reaction volume contained 31 μl cell lysate, 1 μl λ-phosphatase (NEB, 400,000 U/μl), and 8 μl of phosphatase reaction buffer (NEB).

    Article Title: Coordination of microbe–host homeostasis by crosstalk with plant innate immunity
    Article Snippet: Primers and proteins were digested by adding 1 μl of Antarctic phosphatase, 1 μl exonuclease I and 2.44 μl Antarctic phosphatase buffer (New England Biolabs) to 20 μl of the pooled replicate reactions at 37 °C for 30 min, followed by enzyme deactivation at 85 °C for 15 min.

    Polymerase Chain Reaction:

    Article Title: Characterization of the Chlamydomonas reinhardtii phycosphere reveals conserved features of the plant microbiota
    Article Snippet: .. Afterwards, single-stranded DNA and proteins were digested by adding 1 μL of Antarctic phosphatase, 1 μL Exonuclease I and 2.44 μL Antarctic Phosphatase buffer (New England BioLabs GmbH, Frankfurt, Germany) to 20 μl of the pooled PCR product. ..

    Article Title: A fungal powdery mildew pathogen induces extensive local and marginal systemic changes in the Arabidopsis thaliana microbiota
    Article Snippet: .. Afterwards, single-stranded DNA and proteins were digested by adding 1 μL of Antarctic phosphatase, 1 μL xonuclease I and 2.44 μL Antarctic Phosphatase buffer (New England BioLabs GmbH, Frankfurt, Germany) to 20 μL of the pooled PCR product. ..

    Incubation:

    Article Title: Mapping the functional landscape of the receptor binding domain of T7 bacteriophage by deep mutational scanning
    Article Snippet: .. A 5 μl 10× CutSmart, 2 μl BsaI, ~1177 ng backbone, dH2 O to 50 μl was mixed and incubated at 37°C for 2 hr, after which 1 μl additional BsaI, 2 μl Antarctic Phosphatase, 5.89 μl 10× Antarctic Phosphatase buffer was spiked into reaction. ..

    Protein Kinase Assay:

    Article Title: SILAKin: A novel high throughput SILAC and mass spectrometry-based assay to identify the substratome of kinases secreted by pathogens
    Article Snippet: .. Protein kinase assay and phosphatase treatment For phosphatase treatment, 500 μg of “heavy” or “light” THP-1 protein extract were dephosphorylated with 50 U of Antarctic phosphatase (NEB) in Antarctic phosphatase buffer (NEB) for 30 min at 37 °C. ..

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  • 99
    New England Biolabs calf intestinal alkaline phosphatase
    Upon the completion of the V H and V L libraries, the scFv libraries were constructed via V gene shuffling. The V L gene repertoire was cut out from the V L libraries using NotI enzyme in combination with either AscI or SalI enzyme, and then cloned into the V H library to build the scFv libraries with the two different linkers. CIP stands for <t>calf</t> <t>intestinal</t> <t>alkaline</t> <t>phosphatase.</t>
    Calf Intestinal Alkaline Phosphatase, 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/calf intestinal alkaline phosphatase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    99
    New England Biolabs lambda phosphatase
    CKIɛ and CKIδ bind to and phosphorylate mPer proteins specifically. (A) Luciferase (control), mPer1, mPer2, mPer3, mCry1, mCry2, and mBMAL1 were separately synthesized in vitro by using TNT T7 Coupled Reticulocyte Lysate System (right panel) and then mixed with in vitro-synthesized HA-tagged CKIɛ or CKIɛ(KR) and incubated. Each of these mixtures was subjected to immunoprecipitation with anti-HA antibody (12CA5). After resolution by SDS-PAGE, immunoprecipitates were detected by a Bio-Rad phosphorimager (left panel). (B) Myc-tagged mPer1, mPer2, mPer3, mCry1, mCry2, or mBMAL1 was expressed in COS7 cells. Each of the cell extracts was incubated with GST, GST-fused CKIɛ, or GST-fused CKIɛ(KR) (expressed in E. coli , and purified) and glutathione-Sepharose 4B. The beads were then washed with the incubation buffer. After resolution by SDS-PAGE, proteins were analyzed by immunoblotting with anti-Myc antibody (A-14). (C) Myc-tagged mPer1, mPer2, mPer3, mCry1, mCry2, or mBMAL1 was expressed in COS7 cells. The cell extracts were subjected to immunoprecipitation with anti-Myc antibody (9E10). The washed beads were mixed with a kinase reaction buffer supplemented with GST-fused CKIɛ or GST-fused CKIɛ(KR), as well as [γ- 32 P]ATP, and incubated for 30 min. After resolution by SDS-PAGE, substrate phosphorylation was detected by a Bio-Rad phosphorimager. (D) Myc-tagged mPer1, mPer2, mPer3, mCry1, mCry2, or mBMAL1 was expressed in COS7 cells, together with HA-tagged CKIɛ, CKIɛΔC, CKIδ, or CKIɛ(KR). The cell lysates were subjected to immunoprecipitation with anti-HA antibody (12CA5). After resolution by SDS-PAGE, immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody (A-14). (E) Myc-tagged mPer1, mPer2, or mPer3 was expressed in COS7 cells, together with or without CKIδ. The cell lysates were subjected to immunoprecipitation with anti-Myc antibody (9E10), and the washed beads were resuspended in phosphatase buffer and incubated with or without (mock) 40 U of purified <t>lambda</t> phosphatase for 30 min. After resolution by SDS-PAGE, the immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody (A-14).
    Lambda Phosphatase, 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
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    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    lambda phosphatase - by Bioz Stars, 2021-09
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    Upon the completion of the V H and V L libraries, the scFv libraries were constructed via V gene shuffling. The V L gene repertoire was cut out from the V L libraries using NotI enzyme in combination with either AscI or SalI enzyme, and then cloned into the V H library to build the scFv libraries with the two different linkers. CIP stands for calf intestinal alkaline phosphatase.

    Journal: Protein Engineering, Design and Selection

    Article Title: A fully human scFv phage display library for rapid antibody fragment reformatting

    doi: 10.1093/protein/gzv024

    Figure Lengend Snippet: Upon the completion of the V H and V L libraries, the scFv libraries were constructed via V gene shuffling. The V L gene repertoire was cut out from the V L libraries using NotI enzyme in combination with either AscI or SalI enzyme, and then cloned into the V H library to build the scFv libraries with the two different linkers. CIP stands for calf intestinal alkaline phosphatase.

    Article Snippet: The 17aa-SSA-linker-pHEN1 vector DNA was extracted from the bacteria with the QIAprep Spin Miniprep Kit (Qiagen, cat#: 27106), and digested with of NcoI and AscI for 6 h before another hour of calf intestinal alkaline phosphatase (New England Biolabs) treatment at 37°C.

    Techniques: Construct, Clone Assay

    Schematic outline of the construction of the V H and V L ). The V genes were PCR-amplified and cloned into 17aa-SSA-linker-pHEN1 and 18aa-SX-linker-pHEN1 vectors to build the V H and V L libraries. CIP stands for calf intestinal alkaline phosphatase.

    Journal: Protein Engineering, Design and Selection

    Article Title: A fully human scFv phage display library for rapid antibody fragment reformatting

    doi: 10.1093/protein/gzv024

    Figure Lengend Snippet: Schematic outline of the construction of the V H and V L ). The V genes were PCR-amplified and cloned into 17aa-SSA-linker-pHEN1 and 18aa-SX-linker-pHEN1 vectors to build the V H and V L libraries. CIP stands for calf intestinal alkaline phosphatase.

    Article Snippet: The 17aa-SSA-linker-pHEN1 vector DNA was extracted from the bacteria with the QIAprep Spin Miniprep Kit (Qiagen, cat#: 27106), and digested with of NcoI and AscI for 6 h before another hour of calf intestinal alkaline phosphatase (New England Biolabs) treatment at 37°C.

    Techniques: Polymerase Chain Reaction, Amplification, Clone Assay

    CKIɛ and CKIδ bind to and phosphorylate mPer proteins specifically. (A) Luciferase (control), mPer1, mPer2, mPer3, mCry1, mCry2, and mBMAL1 were separately synthesized in vitro by using TNT T7 Coupled Reticulocyte Lysate System (right panel) and then mixed with in vitro-synthesized HA-tagged CKIɛ or CKIɛ(KR) and incubated. Each of these mixtures was subjected to immunoprecipitation with anti-HA antibody (12CA5). After resolution by SDS-PAGE, immunoprecipitates were detected by a Bio-Rad phosphorimager (left panel). (B) Myc-tagged mPer1, mPer2, mPer3, mCry1, mCry2, or mBMAL1 was expressed in COS7 cells. Each of the cell extracts was incubated with GST, GST-fused CKIɛ, or GST-fused CKIɛ(KR) (expressed in E. coli , and purified) and glutathione-Sepharose 4B. The beads were then washed with the incubation buffer. After resolution by SDS-PAGE, proteins were analyzed by immunoblotting with anti-Myc antibody (A-14). (C) Myc-tagged mPer1, mPer2, mPer3, mCry1, mCry2, or mBMAL1 was expressed in COS7 cells. The cell extracts were subjected to immunoprecipitation with anti-Myc antibody (9E10). The washed beads were mixed with a kinase reaction buffer supplemented with GST-fused CKIɛ or GST-fused CKIɛ(KR), as well as [γ- 32 P]ATP, and incubated for 30 min. After resolution by SDS-PAGE, substrate phosphorylation was detected by a Bio-Rad phosphorimager. (D) Myc-tagged mPer1, mPer2, mPer3, mCry1, mCry2, or mBMAL1 was expressed in COS7 cells, together with HA-tagged CKIɛ, CKIɛΔC, CKIδ, or CKIɛ(KR). The cell lysates were subjected to immunoprecipitation with anti-HA antibody (12CA5). After resolution by SDS-PAGE, immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody (A-14). (E) Myc-tagged mPer1, mPer2, or mPer3 was expressed in COS7 cells, together with or without CKIδ. The cell lysates were subjected to immunoprecipitation with anti-Myc antibody (9E10), and the washed beads were resuspended in phosphatase buffer and incubated with or without (mock) 40 U of purified lambda phosphatase for 30 min. After resolution by SDS-PAGE, the immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody (A-14).

    Journal: Molecular and Cellular Biology

    Article Title: Control of Intracellular Dynamics of Mammalian Period Proteins by Casein Kinase I ? (CKI?) and CKI? in Cultured Cells

    doi: 10.1128/MCB.22.6.1693-1703.2002

    Figure Lengend Snippet: CKIɛ and CKIδ bind to and phosphorylate mPer proteins specifically. (A) Luciferase (control), mPer1, mPer2, mPer3, mCry1, mCry2, and mBMAL1 were separately synthesized in vitro by using TNT T7 Coupled Reticulocyte Lysate System (right panel) and then mixed with in vitro-synthesized HA-tagged CKIɛ or CKIɛ(KR) and incubated. Each of these mixtures was subjected to immunoprecipitation with anti-HA antibody (12CA5). After resolution by SDS-PAGE, immunoprecipitates were detected by a Bio-Rad phosphorimager (left panel). (B) Myc-tagged mPer1, mPer2, mPer3, mCry1, mCry2, or mBMAL1 was expressed in COS7 cells. Each of the cell extracts was incubated with GST, GST-fused CKIɛ, or GST-fused CKIɛ(KR) (expressed in E. coli , and purified) and glutathione-Sepharose 4B. The beads were then washed with the incubation buffer. After resolution by SDS-PAGE, proteins were analyzed by immunoblotting with anti-Myc antibody (A-14). (C) Myc-tagged mPer1, mPer2, mPer3, mCry1, mCry2, or mBMAL1 was expressed in COS7 cells. The cell extracts were subjected to immunoprecipitation with anti-Myc antibody (9E10). The washed beads were mixed with a kinase reaction buffer supplemented with GST-fused CKIɛ or GST-fused CKIɛ(KR), as well as [γ- 32 P]ATP, and incubated for 30 min. After resolution by SDS-PAGE, substrate phosphorylation was detected by a Bio-Rad phosphorimager. (D) Myc-tagged mPer1, mPer2, mPer3, mCry1, mCry2, or mBMAL1 was expressed in COS7 cells, together with HA-tagged CKIɛ, CKIɛΔC, CKIδ, or CKIɛ(KR). The cell lysates were subjected to immunoprecipitation with anti-HA antibody (12CA5). After resolution by SDS-PAGE, immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody (A-14). (E) Myc-tagged mPer1, mPer2, or mPer3 was expressed in COS7 cells, together with or without CKIδ. The cell lysates were subjected to immunoprecipitation with anti-Myc antibody (9E10), and the washed beads were resuspended in phosphatase buffer and incubated with or without (mock) 40 U of purified lambda phosphatase for 30 min. After resolution by SDS-PAGE, the immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody (A-14).

    Article Snippet: The beads were washed and resuspended in phosphatase buffer (50 mM Tris-HCl, 0.1 mM EDTA, 5 mM DTT, 0.01% Brij 35, 2 mM MnCl2 ; pH 7.0) and then incubated with 40 U of purified lambda phosphatase (New England Biolabs) for 30 min at 30°C.

    Techniques: Luciferase, Synthesized, In Vitro, Incubation, Immunoprecipitation, SDS Page, Purification

    Identification of potential CKIɛ phosphorylation sites on mPer3. (A) Amino acid sequences of the mammalian Per family. Amino acid identities and similarities are indicated by dark gray and light gray boxes, respectively. The conserved serine-threonine residues are indicated by open circles. (B) PCR-generated alanine mutations were made in the mPer3 conserved serine-threonine cluster and compared with the wild-type (WT) mPer3 sequence. (C and D) Wild-type (WT) mPer3 or each of various mutants of mPer3 (0.7 μg of DNA) was expressed in COS7 cells, together with or without CKIɛ (0.3 μg of DNA). The cell extracts were subjected to immunoblotting with anti-Myc antibody (A-14). Equal amounts of proteins were loaded. The electrophoretic retardation of the mPer3 protein bands results from phosphorylation. (E) Myc-tagged mut7 was expressed in COS7 cells, together with or without CKIɛ. The cell lysates were subjected to immunoprecipitation with anti-Myc antibody (9E10) and incubated with or without (mock) purified lambda phosphatase. The immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody (A-14).

    Journal: Molecular and Cellular Biology

    Article Title: Control of Intracellular Dynamics of Mammalian Period Proteins by Casein Kinase I ? (CKI?) and CKI? in Cultured Cells

    doi: 10.1128/MCB.22.6.1693-1703.2002

    Figure Lengend Snippet: Identification of potential CKIɛ phosphorylation sites on mPer3. (A) Amino acid sequences of the mammalian Per family. Amino acid identities and similarities are indicated by dark gray and light gray boxes, respectively. The conserved serine-threonine residues are indicated by open circles. (B) PCR-generated alanine mutations were made in the mPer3 conserved serine-threonine cluster and compared with the wild-type (WT) mPer3 sequence. (C and D) Wild-type (WT) mPer3 or each of various mutants of mPer3 (0.7 μg of DNA) was expressed in COS7 cells, together with or without CKIɛ (0.3 μg of DNA). The cell extracts were subjected to immunoblotting with anti-Myc antibody (A-14). Equal amounts of proteins were loaded. The electrophoretic retardation of the mPer3 protein bands results from phosphorylation. (E) Myc-tagged mut7 was expressed in COS7 cells, together with or without CKIɛ. The cell lysates were subjected to immunoprecipitation with anti-Myc antibody (9E10) and incubated with or without (mock) purified lambda phosphatase. The immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody (A-14).

    Article Snippet: The beads were washed and resuspended in phosphatase buffer (50 mM Tris-HCl, 0.1 mM EDTA, 5 mM DTT, 0.01% Brij 35, 2 mM MnCl2 ; pH 7.0) and then incubated with 40 U of purified lambda phosphatase (New England Biolabs) for 30 min at 30°C.

    Techniques: Polymerase Chain Reaction, Generated, Sequencing, Immunoprecipitation, Incubation, Purification

    H2AS15ph is locally induced upon DNA double strand break formation and spread over several kilobases. (A) H2A-S15ph signal increased when yeast cells are treated with DNA damaging agent, MMS. Western blot analysis was performed with 300ng of native chromatin purified from yeast cells treated with DMSO (untreated control) and MMS. Anti-H2AS129ph was used as positive control for the MMS treatment. Antibodies against histone H2B and H4 were used as loading control. (B) Anti-H2AS15ph is specific for recognizing a phosphorylation mark on H2A. Western blot analysis was performed with 300ng of native chromatin purified form MMS treated yeast cells after incubation with or without lambda protein phosphatase (λPP). Lane with λPP treatment showed a significant decrease in signal for H2A-S15ph. Histone H4 was used as loading control. (C) H2AS15ph spreads on both sides of the HO endonuclease induced single DSB at MAT locus. Signal for H2AS15ph showed enrichment after the induction of the DSB in wild type but not in H2A-S15A . ChIP-qPCR was performed as described in Figure 1D . Fold enrichment was calculated as ratio of %IP/ input normalized on total H4 for histone occupancy at indicated loci around DSB at MAT locus to the signal at negative-control locus intergenicV. (D, E) Kinetics of H2S15ph and H2AS129ph accumulation after the induction of the HO-DSB. ChIP samples were analyzed after times 0, 30min, 1hr and 3hrs. Anti-H4 signal was used to normalize for histone occupancy. ChIP-qPCR was performed as described in Figure 1D .

    Journal: bioRxiv

    Article Title: DNA damage-induced phosphorylation of histone H2A at serine 15 is linked to DNA end resection

    doi: 10.1101/2021.02.08.430376

    Figure Lengend Snippet: H2AS15ph is locally induced upon DNA double strand break formation and spread over several kilobases. (A) H2A-S15ph signal increased when yeast cells are treated with DNA damaging agent, MMS. Western blot analysis was performed with 300ng of native chromatin purified from yeast cells treated with DMSO (untreated control) and MMS. Anti-H2AS129ph was used as positive control for the MMS treatment. Antibodies against histone H2B and H4 were used as loading control. (B) Anti-H2AS15ph is specific for recognizing a phosphorylation mark on H2A. Western blot analysis was performed with 300ng of native chromatin purified form MMS treated yeast cells after incubation with or without lambda protein phosphatase (λPP). Lane with λPP treatment showed a significant decrease in signal for H2A-S15ph. Histone H4 was used as loading control. (C) H2AS15ph spreads on both sides of the HO endonuclease induced single DSB at MAT locus. Signal for H2AS15ph showed enrichment after the induction of the DSB in wild type but not in H2A-S15A . ChIP-qPCR was performed as described in Figure 1D . Fold enrichment was calculated as ratio of %IP/ input normalized on total H4 for histone occupancy at indicated loci around DSB at MAT locus to the signal at negative-control locus intergenicV. (D, E) Kinetics of H2S15ph and H2AS129ph accumulation after the induction of the HO-DSB. ChIP samples were analyzed after times 0, 30min, 1hr and 3hrs. Anti-H4 signal was used to normalize for histone occupancy. ChIP-qPCR was performed as described in Figure 1D .

    Article Snippet: Lambda protein phosphatase (λPP) (NEB) kit was used.

    Techniques: Western Blot, Purification, Positive Control, Incubation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    Mms4 undergoes cell cycle-dependent phosphorylation. ( A ) MUS81-HA and HA-MMS4 cells were blocked in G1 using α factor and then released from the block and monitored for approximately two cell cycles. The cells were collected at the indicated time points and the DNA content was determined by flow cytometry. ( B ) Percentage of budded and binucleated cells throughout the experiment. ( C ) Immunoblot analysis of the Mus81-HA and HA-Mms4 proteins throughout the experiment. A Pounceau S stained membrane coincident with Mus81/Mms4 migration was used as a loading control. ( D ) HA-MMS4 cells were blocked in G1 using α factor and then released into S-phase either in the absence or the presence of nocodazole in the medium. The cells were collected at the indicated time points, and the DNA content was determined by flow cytometry. ( E ) Percentage of budded and binucleated cells at each time point. ( F ) Immunoblot analysis of HA-Mms4 during the course of the experiment. ( G ) Phosphatase assay. HA-Mms4 was immunoprecipitated from extracts obtained from nocodazole-arrested cells. The protein was then incubated with or without λ-phosphatase and with λ-phosphatase plus phosphatase inhibitors prior to immunoblot analysis. Phosphorylated Mms4 is indicated as ‘Mms4-P’.

    Journal: Nucleic Acids Research

    Article Title: Cell cycle-dependent regulation of the nuclease activity of Mus81-Eme1/Mms4

    doi: 10.1093/nar/gks599

    Figure Lengend Snippet: Mms4 undergoes cell cycle-dependent phosphorylation. ( A ) MUS81-HA and HA-MMS4 cells were blocked in G1 using α factor and then released from the block and monitored for approximately two cell cycles. The cells were collected at the indicated time points and the DNA content was determined by flow cytometry. ( B ) Percentage of budded and binucleated cells throughout the experiment. ( C ) Immunoblot analysis of the Mus81-HA and HA-Mms4 proteins throughout the experiment. A Pounceau S stained membrane coincident with Mus81/Mms4 migration was used as a loading control. ( D ) HA-MMS4 cells were blocked in G1 using α factor and then released into S-phase either in the absence or the presence of nocodazole in the medium. The cells were collected at the indicated time points, and the DNA content was determined by flow cytometry. ( E ) Percentage of budded and binucleated cells at each time point. ( F ) Immunoblot analysis of HA-Mms4 during the course of the experiment. ( G ) Phosphatase assay. HA-Mms4 was immunoprecipitated from extracts obtained from nocodazole-arrested cells. The protein was then incubated with or without λ-phosphatase and with λ-phosphatase plus phosphatase inhibitors prior to immunoblot analysis. Phosphorylated Mms4 is indicated as ‘Mms4-P’.

    Article Snippet: The reactions were carried out at 30°C for 30 min using 8 U of λ-phosphatase (New England Biolabs) in PMP buffer plus 1 mM MnCl2 , 2 mM Na3 VO4 , 20 mM NaF and 2 mM sodium glycerophosphate, and stopped with Laemmli buffer.

    Techniques: Blocking Assay, Flow Cytometry, Cytometry, Staining, Migration, Phosphatase Assay, Immunoprecipitation, Incubation