vitro phosphorylated mbp hd fusion protein Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore polyvinylidene difluoride pvdf membrane
    Phosphorylation of <t>Csx/Nkx2.5</t> by CKII within the homeodomain. (A) In-gel kinase assays. NIH 3T3 cell nuclear extracts (25 μg) were separated in a gel containing either the homeodomain-MBP fusion protein (HD), the full-length Csx/Nkx2.5-MBP fusion protein (Csx), or MBP. After denaturation and renaturation, gels were incubated in kinase buffer containing [γ- 32 P]ATP. One kinase with a molecular mass of ∼40 kDa (arrowheads) phosphorylated the Csx/Nkx2.5 homeodomain, Csx/Nkx2.5 (lane 1), and the full-length Csx/Nkx2.5 (lane 2), but not MBP (lane 3). (B) Schematic representation of Csx/Nkx2.5 mutants carrying the consensus CKII sites and serine-to-alanine substitutions. (C) In vitro CKII phosphorylation of Csx/Nkx2.5. Three different Csx/Nkx2.5 fusion proteins described in panel B were incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of purified CKII. Each sample was subjected to SDS-PAGE and transferred to a <t>PVDF</t> membrane. Autoradiography (upper panel) revealed that CKII phosphorylated the full-length Csx/Nkx2.5 (lane 1) and the homeodomain (lane 3) but neither the C-terminally deleted mutant (lane 5) nor MBP (lane 7). Loaded fusion proteins are shown by Western blotting of the same PVDF membrane using anti-MBP antibody (lower panel). (D) Serine 163 was mutated into alanine (Csx/Nkx2.5 163S-A ) and the kinase assay was performed. Lane 1 contains the wild-type Csx/Nkx2.5 and lane 2 contains the mutant Csx/Nkx2.5 163S-A . Equal amounts of proteins were subjected to the kinase reaction as shown in the MBP Western blot (lower panel).
    Polyvinylidene Difluoride Pvdf Membrane, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12062 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyvinylidene difluoride pvdf membrane/product/Millipore
    Average 99 stars, based on 12062 article reviews
    Price from $9.99 to $1999.99
    polyvinylidene difluoride pvdf membrane - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    TaKaRa in fusion hd cloning
    Phosphorylation of <t>Csx/Nkx2.5</t> by CKII within the homeodomain. (A) In-gel kinase assays. NIH 3T3 cell nuclear extracts (25 μg) were separated in a gel containing either the homeodomain-MBP fusion protein (HD), the full-length Csx/Nkx2.5-MBP fusion protein (Csx), or MBP. After denaturation and renaturation, gels were incubated in kinase buffer containing [γ- 32 P]ATP. One kinase with a molecular mass of ∼40 kDa (arrowheads) phosphorylated the Csx/Nkx2.5 homeodomain, Csx/Nkx2.5 (lane 1), and the full-length Csx/Nkx2.5 (lane 2), but not MBP (lane 3). (B) Schematic representation of Csx/Nkx2.5 mutants carrying the consensus CKII sites and serine-to-alanine substitutions. (C) In vitro CKII phosphorylation of Csx/Nkx2.5. Three different Csx/Nkx2.5 fusion proteins described in panel B were incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of purified CKII. Each sample was subjected to SDS-PAGE and transferred to a <t>PVDF</t> membrane. Autoradiography (upper panel) revealed that CKII phosphorylated the full-length Csx/Nkx2.5 (lane 1) and the homeodomain (lane 3) but neither the C-terminally deleted mutant (lane 5) nor MBP (lane 7). Loaded fusion proteins are shown by Western blotting of the same PVDF membrane using anti-MBP antibody (lower panel). (D) Serine 163 was mutated into alanine (Csx/Nkx2.5 163S-A ) and the kinase assay was performed. Lane 1 contains the wild-type Csx/Nkx2.5 and lane 2 contains the mutant Csx/Nkx2.5 163S-A . Equal amounts of proteins were subjected to the kinase reaction as shown in the MBP Western blot (lower panel).
    In Fusion Hd Cloning, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 740 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/in fusion hd cloning/product/TaKaRa
    Average 99 stars, based on 740 article reviews
    Price from $9.99 to $1999.99
    in fusion hd cloning - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Phosphorylation of Csx/Nkx2.5 by CKII within the homeodomain. (A) In-gel kinase assays. NIH 3T3 cell nuclear extracts (25 μg) were separated in a gel containing either the homeodomain-MBP fusion protein (HD), the full-length Csx/Nkx2.5-MBP fusion protein (Csx), or MBP. After denaturation and renaturation, gels were incubated in kinase buffer containing [γ- 32 P]ATP. One kinase with a molecular mass of ∼40 kDa (arrowheads) phosphorylated the Csx/Nkx2.5 homeodomain, Csx/Nkx2.5 (lane 1), and the full-length Csx/Nkx2.5 (lane 2), but not MBP (lane 3). (B) Schematic representation of Csx/Nkx2.5 mutants carrying the consensus CKII sites and serine-to-alanine substitutions. (C) In vitro CKII phosphorylation of Csx/Nkx2.5. Three different Csx/Nkx2.5 fusion proteins described in panel B were incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of purified CKII. Each sample was subjected to SDS-PAGE and transferred to a PVDF membrane. Autoradiography (upper panel) revealed that CKII phosphorylated the full-length Csx/Nkx2.5 (lane 1) and the homeodomain (lane 3) but neither the C-terminally deleted mutant (lane 5) nor MBP (lane 7). Loaded fusion proteins are shown by Western blotting of the same PVDF membrane using anti-MBP antibody (lower panel). (D) Serine 163 was mutated into alanine (Csx/Nkx2.5 163S-A ) and the kinase assay was performed. Lane 1 contains the wild-type Csx/Nkx2.5 and lane 2 contains the mutant Csx/Nkx2.5 163S-A . Equal amounts of proteins were subjected to the kinase reaction as shown in the MBP Western blot (lower panel).

    Journal: Molecular and Cellular Biology

    Article Title: Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

    doi:

    Figure Lengend Snippet: Phosphorylation of Csx/Nkx2.5 by CKII within the homeodomain. (A) In-gel kinase assays. NIH 3T3 cell nuclear extracts (25 μg) were separated in a gel containing either the homeodomain-MBP fusion protein (HD), the full-length Csx/Nkx2.5-MBP fusion protein (Csx), or MBP. After denaturation and renaturation, gels were incubated in kinase buffer containing [γ- 32 P]ATP. One kinase with a molecular mass of ∼40 kDa (arrowheads) phosphorylated the Csx/Nkx2.5 homeodomain, Csx/Nkx2.5 (lane 1), and the full-length Csx/Nkx2.5 (lane 2), but not MBP (lane 3). (B) Schematic representation of Csx/Nkx2.5 mutants carrying the consensus CKII sites and serine-to-alanine substitutions. (C) In vitro CKII phosphorylation of Csx/Nkx2.5. Three different Csx/Nkx2.5 fusion proteins described in panel B were incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of purified CKII. Each sample was subjected to SDS-PAGE and transferred to a PVDF membrane. Autoradiography (upper panel) revealed that CKII phosphorylated the full-length Csx/Nkx2.5 (lane 1) and the homeodomain (lane 3) but neither the C-terminally deleted mutant (lane 5) nor MBP (lane 7). Loaded fusion proteins are shown by Western blotting of the same PVDF membrane using anti-MBP antibody (lower panel). (D) Serine 163 was mutated into alanine (Csx/Nkx2.5 163S-A ) and the kinase assay was performed. Lane 1 contains the wild-type Csx/Nkx2.5 and lane 2 contains the mutant Csx/Nkx2.5 163S-A . Equal amounts of proteins were subjected to the kinase reaction as shown in the MBP Western blot (lower panel).

    Article Snippet: Immunoprecipitants of in vivo-labeled Csx/Nkx2.5 or in vitro-phosphorylated MBP-HD fusion protein were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) after SDS-PAGE and then autoradiographed.

    Techniques: Incubation, In Vitro, Purification, SDS Page, Autoradiography, Mutagenesis, Western Blot, Kinase Assay

    Phosphopeptide and phosphoamino acid analysis of Csx/Nkx2.5. (A) Tryptic phosphopeptide mapping of wild-type Csx/Nkx2.5 (a), CKII mutant (b), and in vitro CKII-phosphorylated MBP-homeodomain protein (c). Metabolically 32 P-labeled Csx/Nkx2.5 and the CKII mutant (Csx/Nkx2.5 163S-A ) were immunoprecipitated with anti-Csx/Nkx2.5 MAb (2D10), resolved by SDS-PAGE, transferred to a PVDF membrane, and autoradiographed. In vitro CKII-phosphorylated MBP-homeodomain protein was also electrophoresed and autoradiographed. These protein bands were cut out, digested with trypsin, and resolved on TLC plates by electrophoresis in the first dimension and chromatography in the second dimension. In vitro CKII-phosphorylated peptide (c [arrow]) which corresponded to peptide 1 in the wild type (a [arrow]), was markedly reduced in the CKII mutant (b). Arrowheads, sample loaded points; X, lysine marker. (B) Phosphoamino acid analysis of wild-type Csx/Nkx2.5 showed Csx/Nkx2.5 is phosphorylated predominantly in S and weakly in T.

    Journal: Molecular and Cellular Biology

    Article Title: Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

    doi:

    Figure Lengend Snippet: Phosphopeptide and phosphoamino acid analysis of Csx/Nkx2.5. (A) Tryptic phosphopeptide mapping of wild-type Csx/Nkx2.5 (a), CKII mutant (b), and in vitro CKII-phosphorylated MBP-homeodomain protein (c). Metabolically 32 P-labeled Csx/Nkx2.5 and the CKII mutant (Csx/Nkx2.5 163S-A ) were immunoprecipitated with anti-Csx/Nkx2.5 MAb (2D10), resolved by SDS-PAGE, transferred to a PVDF membrane, and autoradiographed. In vitro CKII-phosphorylated MBP-homeodomain protein was also electrophoresed and autoradiographed. These protein bands were cut out, digested with trypsin, and resolved on TLC plates by electrophoresis in the first dimension and chromatography in the second dimension. In vitro CKII-phosphorylated peptide (c [arrow]) which corresponded to peptide 1 in the wild type (a [arrow]), was markedly reduced in the CKII mutant (b). Arrowheads, sample loaded points; X, lysine marker. (B) Phosphoamino acid analysis of wild-type Csx/Nkx2.5 showed Csx/Nkx2.5 is phosphorylated predominantly in S and weakly in T.

    Article Snippet: Immunoprecipitants of in vivo-labeled Csx/Nkx2.5 or in vitro-phosphorylated MBP-HD fusion protein were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) after SDS-PAGE and then autoradiographed.

    Techniques: Phosphoamino Acid Analysis, Mutagenesis, In Vitro, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Thin Layer Chromatography, Electrophoresis, Chromatography, Marker