heparinase iii treatment  (Millipore)


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  • 98
    Name:
    Heparinase I and III Blend from Flavobacterium heparinum
    Description:
    Heparinase is an inducible non extracellular heparin degrading enzyme Three types of heparinises are produced by Flavobacterium heparinum and contains specific sequences of heparin
    Catalog Number:
    h3917
    Price:
    None
    Applications:
    Heparinase I and III may be used for the study of heparin production during fermentation and specific activity of heparinise.
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    Structured Review

    Millipore heparinase iii treatment
    The interaction of LRRTM4 with GPC4 is direct and requires HS (A) Direct interaction of recombinant His-tagged ecto-LRRTM4 with GPC4-Fc. Fc, GPC4-Fc, Nrx1ß(−S4)-Fc or LPHN3-Fc were mixed with His-LRRTM4, precipitated and analyzed by Western blot. His-LRRTM4 binds to GPC4-Fc and Nrx1ß(−S4)-Fc, but not to Fc or LPHN3-Fc. (B) LRRTM4 bound to Nrx1ß(−S4) cannot simultaneously bind to GPC4. Recombinant HA-GPC4, Nrx1ß(−S4)-Fc and His-LRRTM4 or His-FLRT3 were mixed and precipitated with protein A/G agarose. Proteins bound to Nrx1ß(−S4)-Fc were analyzed with His and HA antibodies. Blot shows full-length GPC4. (C) LRRTM4 bound to GPC4 cannot simultaneously bind to Nrx1ß(−S4). HA-GPC4, Nrx1ß(−S4)-Fc and His-LRRTM4 or His-FLRT3 were mixed and precipitated with HA affinity matrix. Proteins bound to HA-GPC4 were analyzed with His and Fc antibodies. (D) Cell surface binding assays. LRRTM4-Fc (red) binding to HA-GPC4 (green) expressing HEK293T cells is strongly reduced in the presence of 0.5 mg/ml HS or following treatment with 1 U/ml <t>heparinase</t> (hep) <t>III.</t> HS and hepIII abolish background binding of LRRTM4-Fc to cells expressing vector alone. (E) Quantification of assays in (D). Bar graph shows mean ± SEM; a.u., arbitrary units. Only LRRTM4-Fc binding to HA-GPC4 + vehicle was significantly above the pDisplay + vehicle control condition (***p
    Heparinase is an inducible non extracellular heparin degrading enzyme Three types of heparinises are produced by Flavobacterium heparinum and contains specific sequences of heparin
    https://www.bioz.com/result/heparinase iii treatment/product/Millipore
    Average 98 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    heparinase iii treatment - by Bioz Stars, 2020-11
    98/100 stars

    Related Products / Commonly Used Together

    heparinase iii
    hippocampal neurons

    Images

    1) Product Images from "Unbiased Discovery of Glypican as a Novel Receptor for LRRTM4 in Regulating Excitatory Synapse Development"

    Article Title: Unbiased Discovery of Glypican as a Novel Receptor for LRRTM4 in Regulating Excitatory Synapse Development

    Journal: Neuron

    doi: 10.1016/j.neuron.2013.06.049

    The interaction of LRRTM4 with GPC4 is direct and requires HS (A) Direct interaction of recombinant His-tagged ecto-LRRTM4 with GPC4-Fc. Fc, GPC4-Fc, Nrx1ß(−S4)-Fc or LPHN3-Fc were mixed with His-LRRTM4, precipitated and analyzed by Western blot. His-LRRTM4 binds to GPC4-Fc and Nrx1ß(−S4)-Fc, but not to Fc or LPHN3-Fc. (B) LRRTM4 bound to Nrx1ß(−S4) cannot simultaneously bind to GPC4. Recombinant HA-GPC4, Nrx1ß(−S4)-Fc and His-LRRTM4 or His-FLRT3 were mixed and precipitated with protein A/G agarose. Proteins bound to Nrx1ß(−S4)-Fc were analyzed with His and HA antibodies. Blot shows full-length GPC4. (C) LRRTM4 bound to GPC4 cannot simultaneously bind to Nrx1ß(−S4). HA-GPC4, Nrx1ß(−S4)-Fc and His-LRRTM4 or His-FLRT3 were mixed and precipitated with HA affinity matrix. Proteins bound to HA-GPC4 were analyzed with His and Fc antibodies. (D) Cell surface binding assays. LRRTM4-Fc (red) binding to HA-GPC4 (green) expressing HEK293T cells is strongly reduced in the presence of 0.5 mg/ml HS or following treatment with 1 U/ml heparinase (hep) III. HS and hepIII abolish background binding of LRRTM4-Fc to cells expressing vector alone. (E) Quantification of assays in (D). Bar graph shows mean ± SEM; a.u., arbitrary units. Only LRRTM4-Fc binding to HA-GPC4 + vehicle was significantly above the pDisplay + vehicle control condition (***p
    Figure Legend Snippet: The interaction of LRRTM4 with GPC4 is direct and requires HS (A) Direct interaction of recombinant His-tagged ecto-LRRTM4 with GPC4-Fc. Fc, GPC4-Fc, Nrx1ß(−S4)-Fc or LPHN3-Fc were mixed with His-LRRTM4, precipitated and analyzed by Western blot. His-LRRTM4 binds to GPC4-Fc and Nrx1ß(−S4)-Fc, but not to Fc or LPHN3-Fc. (B) LRRTM4 bound to Nrx1ß(−S4) cannot simultaneously bind to GPC4. Recombinant HA-GPC4, Nrx1ß(−S4)-Fc and His-LRRTM4 or His-FLRT3 were mixed and precipitated with protein A/G agarose. Proteins bound to Nrx1ß(−S4)-Fc were analyzed with His and HA antibodies. Blot shows full-length GPC4. (C) LRRTM4 bound to GPC4 cannot simultaneously bind to Nrx1ß(−S4). HA-GPC4, Nrx1ß(−S4)-Fc and His-LRRTM4 or His-FLRT3 were mixed and precipitated with HA affinity matrix. Proteins bound to HA-GPC4 were analyzed with His and Fc antibodies. (D) Cell surface binding assays. LRRTM4-Fc (red) binding to HA-GPC4 (green) expressing HEK293T cells is strongly reduced in the presence of 0.5 mg/ml HS or following treatment with 1 U/ml heparinase (hep) III. HS and hepIII abolish background binding of LRRTM4-Fc to cells expressing vector alone. (E) Quantification of assays in (D). Bar graph shows mean ± SEM; a.u., arbitrary units. Only LRRTM4-Fc binding to HA-GPC4 + vehicle was significantly above the pDisplay + vehicle control condition (***p

    Techniques Used: Recombinant, Western Blot, Binding Assay, Expressing, Plasmid Preparation

    2) Product Images from "An input-specific orphan receptor GPR158-HSPG interaction organizes hippocampal mossy fiber-CA3 synapses"

    Article Title: An input-specific orphan receptor GPR158-HSPG interaction organizes hippocampal mossy fiber-CA3 synapses

    Journal: Neuron

    doi: 10.1016/j.neuron.2018.08.038

    GPR158 is a heparan sulfate-dependent GPC4 binding partner (A) Proteomic workflow for the identification of GPC4-interacting proteins. (B) Identification of GPR158 as a GPC4 interactor by tandem mass spectrometry. GPC4-Fc protein was used as bait and synaptosome extract from P21 rat brains as prey. Graph shows summed peptide and spectral counts for all surface proteins after Fc background subtraction (n = 3 independent experiments). (C) Freqency of detection of peptides (spectral count) for all proteins identified in two independent GPC4-Fc affinity purification experiments after Fc background subtraction. (D) GPR158 domain organization. LRD, Leucine-Rich Domain; EGF-like, EGF-like Ca 2+ binding motif. Yellow region, RGS7-binding site. (E) Cell-surface binding assays in HEK293T cells. GPC4-Fc (red), but not GPC4 ΔGAG-Fc, binds the GPR158 ectodomain (green) expressed on the cell membrane. LRRTM4 and EGFP serve as positive and negative controls, respectively. (F) Pulldown assay in HEK293T cells. GPC4-Fc, but not Fc alone, binds GPR158. HS removal by mutagenesis (GPC4 ΔGAG-Fc) or Heparinase III treatment (GPC4-Fc/HepIII) abolishes the interaction. (G) GPC4 and GPR158 interact in trans . GPC4 co-immunoprecipitates with GPR158 following expression in separately transfected and co-cultured HEK293T cells. 65 kDa band represents full-length GPC4; 40 kDa band the N-terminal proteolytic fragment. IgG serves as negative control. (H) Pulldown assay in hippocampal lysate. GPR158-Fc, but not Fc alone, binds endogenous GPC4. Scale bar in (E) 10 µm. .
    Figure Legend Snippet: GPR158 is a heparan sulfate-dependent GPC4 binding partner (A) Proteomic workflow for the identification of GPC4-interacting proteins. (B) Identification of GPR158 as a GPC4 interactor by tandem mass spectrometry. GPC4-Fc protein was used as bait and synaptosome extract from P21 rat brains as prey. Graph shows summed peptide and spectral counts for all surface proteins after Fc background subtraction (n = 3 independent experiments). (C) Freqency of detection of peptides (spectral count) for all proteins identified in two independent GPC4-Fc affinity purification experiments after Fc background subtraction. (D) GPR158 domain organization. LRD, Leucine-Rich Domain; EGF-like, EGF-like Ca 2+ binding motif. Yellow region, RGS7-binding site. (E) Cell-surface binding assays in HEK293T cells. GPC4-Fc (red), but not GPC4 ΔGAG-Fc, binds the GPR158 ectodomain (green) expressed on the cell membrane. LRRTM4 and EGFP serve as positive and negative controls, respectively. (F) Pulldown assay in HEK293T cells. GPC4-Fc, but not Fc alone, binds GPR158. HS removal by mutagenesis (GPC4 ΔGAG-Fc) or Heparinase III treatment (GPC4-Fc/HepIII) abolishes the interaction. (G) GPC4 and GPR158 interact in trans . GPC4 co-immunoprecipitates with GPR158 following expression in separately transfected and co-cultured HEK293T cells. 65 kDa band represents full-length GPC4; 40 kDa band the N-terminal proteolytic fragment. IgG serves as negative control. (H) Pulldown assay in hippocampal lysate. GPR158-Fc, but not Fc alone, binds endogenous GPC4. Scale bar in (E) 10 µm. .

    Techniques Used: Binding Assay, Mass Spectrometry, Affinity Purification, Mutagenesis, Expressing, Transfection, Cell Culture, Negative Control

    Related Articles

    Incubation:

    Article Title: Syndecan-Fc Hybrid Molecule as a Potent In Vitro Microbicidal Anti-HIV-1 Agent ▿
    Article Snippet: .. Syndecan-1-Fc was incubated with heparinase I, II, and III (0.3 mIU) (Sigma-Aldrich, St. Louis, MO) for 2 h at 37°C to remove HS chains from the core protein. .. Nunc MaxiSorb eight-well strip plates were coated with anti-syndecan-1 IgG (clone ID4) or control isotype IgG (10 μg/ml) for 16 h at 4°C.

    Recombinant:

    Article Title: Functional Analysis of Corin Protein Domains Required for PCSK6-mediated Activation
    Article Snippet: .. The conditioned medium containing recombinant PCSK6 was added to the cell culture together with heparinase (I and III blend), which removes heparan sulfate chains (Sigma H3917, 0.015, 0.15 and 1.5 U/mL) or chondroitinase ABC, which removes chondroitin sulfate chains (Sigma C3667, 1.5 U/mL). .. Corin protein fragments in the cell lysates were examined by SDS-PAGE and Western blotting.

    Cell Culture:

    Article Title: Functional Analysis of Corin Protein Domains Required for PCSK6-mediated Activation
    Article Snippet: .. The conditioned medium containing recombinant PCSK6 was added to the cell culture together with heparinase (I and III blend), which removes heparan sulfate chains (Sigma H3917, 0.015, 0.15 and 1.5 U/mL) or chondroitinase ABC, which removes chondroitin sulfate chains (Sigma C3667, 1.5 U/mL). .. Corin protein fragments in the cell lysates were examined by SDS-PAGE and Western blotting.

    Positive Control:

    Article Title: Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase
    Article Snippet: .. Commercially available P. heparinus heparinase III (0.5–1-U) (Sigma-Aldrich) was used as a positive control. .. The protein elution buffer was used as a negative control.

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  • 91
    Millipore heparitinase
    Effect of HSGAG modification on FGF-1-induced phosphorylation of FGFR2 and FGFR substrates. (A and B) Receptor autophosphorylation. Cells stably transfected with vector alone (pCEV27) or various forms of FGFR2-B (WT, SAG, ΔA, or 3 Loop) were cultured and exposed to 5 ng of FGF-1/ml. Cell lysates containing 500 μg of protein were immunoprecipitated with anti-FGFR2 antibody and detected by either anti-phosphotyrosine (αPY) (A) or anti-FGFR2 (αFGFR2) (B) antibody as noted on the right. For experiments involving immunoprecipitation of FGFR2, treatments with <t>heparitinase</t> and chondroitinase ABC were performed after immunoprecipitation. The membrane was first used for detection of phosphotyrosine followed by detection of FGFR2 after the antibodies were stripped off. (C) Phosphorylation of receptor kinase substrates. Cell lysates containing 500 μg of protein were immunoprecipitated with αPY and detected by the same antibody. The locations of the molecular mass markers are shown on the left. The arrow denotes the major FGFR substrate of 95 kDa.
    Heparitinase, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparitinase/product/Millipore
    Average 91 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    heparitinase - by Bioz Stars, 2020-11
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    Effect of HSGAG modification on FGF-1-induced phosphorylation of FGFR2 and FGFR substrates. (A and B) Receptor autophosphorylation. Cells stably transfected with vector alone (pCEV27) or various forms of FGFR2-B (WT, SAG, ΔA, or 3 Loop) were cultured and exposed to 5 ng of FGF-1/ml. Cell lysates containing 500 μg of protein were immunoprecipitated with anti-FGFR2 antibody and detected by either anti-phosphotyrosine (αPY) (A) or anti-FGFR2 (αFGFR2) (B) antibody as noted on the right. For experiments involving immunoprecipitation of FGFR2, treatments with heparitinase and chondroitinase ABC were performed after immunoprecipitation. The membrane was first used for detection of phosphotyrosine followed by detection of FGFR2 after the antibodies were stripped off. (C) Phosphorylation of receptor kinase substrates. Cell lysates containing 500 μg of protein were immunoprecipitated with αPY and detected by the same antibody. The locations of the molecular mass markers are shown on the left. The arrow denotes the major FGFR substrate of 95 kDa.

    Journal: Molecular and Cellular Biology

    Article Title: The Acidic Domain and First Immunoglobulin-Like Loop of Fibroblast Growth Factor Receptor 2 Modulate Downstream Signaling through Glycosaminoglycan Modification

    doi:

    Figure Lengend Snippet: Effect of HSGAG modification on FGF-1-induced phosphorylation of FGFR2 and FGFR substrates. (A and B) Receptor autophosphorylation. Cells stably transfected with vector alone (pCEV27) or various forms of FGFR2-B (WT, SAG, ΔA, or 3 Loop) were cultured and exposed to 5 ng of FGF-1/ml. Cell lysates containing 500 μg of protein were immunoprecipitated with anti-FGFR2 antibody and detected by either anti-phosphotyrosine (αPY) (A) or anti-FGFR2 (αFGFR2) (B) antibody as noted on the right. For experiments involving immunoprecipitation of FGFR2, treatments with heparitinase and chondroitinase ABC were performed after immunoprecipitation. The membrane was first used for detection of phosphotyrosine followed by detection of FGFR2 after the antibodies were stripped off. (C) Phosphorylation of receptor kinase substrates. Cell lysates containing 500 μg of protein were immunoprecipitated with αPY and detected by the same antibody. The locations of the molecular mass markers are shown on the left. The arrow denotes the major FGFR substrate of 95 kDa.

    Article Snippet: Cell lysates from affinity-labeling samples were diluted at least 50-fold in a 0.1 M Tris acetate buffer and treated with heparitinase (code 100703; lot no. E95601; Seikagaku America, Rockville, Md.) and/or chondroitinase ABC (code 100332; lot no. KE95702; Seikagaku America) (final concentration, 10 mIU/ml) in 0.1 M Tris acetate buffer, pH 7.3, at 37°C for 1 h. In the case of treatment with heparitinase alone, shark cartilage chondroitin sulfate (catalog no. 2307; Calbiochem) was added to a final concentration of 50 μg/ml to protect the samples from digestion by chondroitinases possibly contaminating the heparitinase preparation.

    Techniques: Modification, Stable Transfection, Transfection, Plasmid Preparation, Cell Culture, Immunoprecipitation