heparitinases  (Millipore)


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    Structured Review

    Millipore heparitinases
    Binding of CXCL12 peptides to HSPG on RA ECs. Rheumatoid arthritis endothelial cells (RA ECs) were incubated with 300 nM biotinylated CXCL12α and, after extensive washing to remove free chemokine, were labeled with fluorescein isothiocyanate-conjugated avidin. Where indicated, RA ECs were simultaneously incubated with soluble sodium heparin (a) or pretreated with <t>heparitinases</t> (b, c) or sodium chlorate (d) . In (b), surface heparan sulfate proteoglycan (HSPG) was detected with 10E4 mAb in RA ECs pretreated or not with heparitinases. (e) RA ECs were incubated with 1 μM non-biotinylated wild-type CXCL12α or 2/6 CXCL12α and labelled with K15C mAb. Filled histograms show isotype control IgG. Results are representative of three to five independent experiments with RA ECs from three different donors. (f) Summary of normalized mea n fluorescence intensity (MFI) data. Error bars show SD. * p
    Heparitinases, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "CXCL12 is displayed by rheumatoid endothelial cells through its basic amino-terminal motif on heparan sulfate proteoglycans"

    Article Title: CXCL12 is displayed by rheumatoid endothelial cells through its basic amino-terminal motif on heparan sulfate proteoglycans

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar1900

    Binding of CXCL12 peptides to HSPG on RA ECs. Rheumatoid arthritis endothelial cells (RA ECs) were incubated with 300 nM biotinylated CXCL12α and, after extensive washing to remove free chemokine, were labeled with fluorescein isothiocyanate-conjugated avidin. Where indicated, RA ECs were simultaneously incubated with soluble sodium heparin (a) or pretreated with heparitinases (b, c) or sodium chlorate (d) . In (b), surface heparan sulfate proteoglycan (HSPG) was detected with 10E4 mAb in RA ECs pretreated or not with heparitinases. (e) RA ECs were incubated with 1 μM non-biotinylated wild-type CXCL12α or 2/6 CXCL12α and labelled with K15C mAb. Filled histograms show isotype control IgG. Results are representative of three to five independent experiments with RA ECs from three different donors. (f) Summary of normalized mea n fluorescence intensity (MFI) data. Error bars show SD. * p
    Figure Legend Snippet: Binding of CXCL12 peptides to HSPG on RA ECs. Rheumatoid arthritis endothelial cells (RA ECs) were incubated with 300 nM biotinylated CXCL12α and, after extensive washing to remove free chemokine, were labeled with fluorescein isothiocyanate-conjugated avidin. Where indicated, RA ECs were simultaneously incubated with soluble sodium heparin (a) or pretreated with heparitinases (b, c) or sodium chlorate (d) . In (b), surface heparan sulfate proteoglycan (HSPG) was detected with 10E4 mAb in RA ECs pretreated or not with heparitinases. (e) RA ECs were incubated with 1 μM non-biotinylated wild-type CXCL12α or 2/6 CXCL12α and labelled with K15C mAb. Filled histograms show isotype control IgG. Results are representative of three to five independent experiments with RA ECs from three different donors. (f) Summary of normalized mea n fluorescence intensity (MFI) data. Error bars show SD. * p

    Techniques Used: Binding Assay, Incubation, Labeling, Avidin-Biotin Assay, Microelectrode Array, Fluorescence

    Related Articles

    Cell Culture:

    Article Title: CXCL12 is displayed by rheumatoid endothelial cells through its basic amino-terminal motif on heparan sulfate proteoglycans
    Article Snippet: .. Removal of surface HSPGs in cultured cells was performed by treatment with a cocktail of 100 mU/ml heparitinases I, II and III each, or 100 mU/ml chondroitin sulfate ABC lyase as a control for 90 minutes at 37°C (Sigma Aldrich Química S.A., Madrid, Spain). .. Where indicated, EC cultures were stimulated with either 25 ng/ml TNF-α or 10 ng/ml LT-α1/β2 (R & D Systems, Inc., Abingdon, Oxon, UK) for 16 hours before CXCL12α binding.

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    Millipore heparitinase
    Heparan sulfate is required for functional presentation of osteoprotegerin on the surface of osteoblasts. ( A ) Structure of osteoprotegerin (OPG) and point mutants generated in this study. The locations of the 3 basic clusters that were mutagenized (C1, C2, C3) are shown. ( B ) Heparin affinity chromatography of OPG mutants. There is essentially no binding of OPG-C3 to heparin, while OPG-C1 and OPG-C2 bind heparin as efficiently as OPG-WT. ( C ) Binding of OPG-WT and OPG-C3 to WT (CHO-K1) and HS-deficient (pgsD-677) CHO cells. Cell surface–bound OPG was quantified as described in Methods. ( D ) Association of endogenous OPG with the surface of WT and Ext1 -null (KO) primary osteoblasts. ( E ) Effect of <t>heparitinase</t> treatment (H’ase) on the association of endogenous OPG with the surface of WT primary osteoblasts. ( F ) Cell surface localization of endogenous OPG in primary human osteoblasts. Cultures of human osteoblasts were double labeled with anti-OPG monoclonal antibody and SiR actin. Control, staining without primary antibody; αOPG, staining with anti-OPG monoclonal antibody; αOPG + H’ase, staining with anti-OPG monoclonal antibody after heparitinase treatment of cells. Scale bar: 10 μm. ( G ) HS binding is necessary for OPG to efficiently inhibit osteoclastogenesis. Cocultures of osteoblasts (OB) and bone marrow macrophages (BM) were prepared in the combination of two OPG forms (OPG-WT [WT] and OPG-C3 [C3]) and osteoblasts of two genotypes (WT and KO), as indicated in the marker table on the right, and emergence of TRAP-positive osteoclasts was quantitated. OPGs were added at 0, 1, 10, 100, and 300 ng/ml, as indicated. Results are shown as the percentage of TRAP-positive cells relative to the total number of cells. Note that little osteoclastogenesis-inhibitory activity is detected in the OPG-WT/ Ext1 -null osteoblast combination (squares) and the OPG-C3/WT osteoblast combination (triangles) at 100 ng/ml, while the OPG-WT/WT osteoblast combination shows a significant inhibitory effects in the range of 1–10 ng/ml and almost complete inhibition at 100 ng/ml (circles). Data represent the mean ± SD (number of cultures tested = 5 in C , 4 in D , and 3 in E and F ). * P
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    Heparan sulfate is required for functional presentation of osteoprotegerin on the surface of osteoblasts. ( A ) Structure of osteoprotegerin (OPG) and point mutants generated in this study. The locations of the 3 basic clusters that were mutagenized (C1, C2, C3) are shown. ( B ) Heparin affinity chromatography of OPG mutants. There is essentially no binding of OPG-C3 to heparin, while OPG-C1 and OPG-C2 bind heparin as efficiently as OPG-WT. ( C ) Binding of OPG-WT and OPG-C3 to WT (CHO-K1) and HS-deficient (pgsD-677) CHO cells. Cell surface–bound OPG was quantified as described in Methods. ( D ) Association of endogenous OPG with the surface of WT and Ext1 -null (KO) primary osteoblasts. ( E ) Effect of heparitinase treatment (H’ase) on the association of endogenous OPG with the surface of WT primary osteoblasts. ( F ) Cell surface localization of endogenous OPG in primary human osteoblasts. Cultures of human osteoblasts were double labeled with anti-OPG monoclonal antibody and SiR actin. Control, staining without primary antibody; αOPG, staining with anti-OPG monoclonal antibody; αOPG + H’ase, staining with anti-OPG monoclonal antibody after heparitinase treatment of cells. Scale bar: 10 μm. ( G ) HS binding is necessary for OPG to efficiently inhibit osteoclastogenesis. Cocultures of osteoblasts (OB) and bone marrow macrophages (BM) were prepared in the combination of two OPG forms (OPG-WT [WT] and OPG-C3 [C3]) and osteoblasts of two genotypes (WT and KO), as indicated in the marker table on the right, and emergence of TRAP-positive osteoclasts was quantitated. OPGs were added at 0, 1, 10, 100, and 300 ng/ml, as indicated. Results are shown as the percentage of TRAP-positive cells relative to the total number of cells. Note that little osteoclastogenesis-inhibitory activity is detected in the OPG-WT/ Ext1 -null osteoblast combination (squares) and the OPG-C3/WT osteoblast combination (triangles) at 100 ng/ml, while the OPG-WT/WT osteoblast combination shows a significant inhibitory effects in the range of 1–10 ng/ml and almost complete inhibition at 100 ng/ml (circles). Data represent the mean ± SD (number of cultures tested = 5 in C , 4 in D , and 3 in E and F ). * P

    Journal: JCI Insight

    Article Title: Osteoblastic heparan sulfate regulates osteoprotegerin function and bone mass

    doi: 10.1172/jci.insight.89624

    Figure Lengend Snippet: Heparan sulfate is required for functional presentation of osteoprotegerin on the surface of osteoblasts. ( A ) Structure of osteoprotegerin (OPG) and point mutants generated in this study. The locations of the 3 basic clusters that were mutagenized (C1, C2, C3) are shown. ( B ) Heparin affinity chromatography of OPG mutants. There is essentially no binding of OPG-C3 to heparin, while OPG-C1 and OPG-C2 bind heparin as efficiently as OPG-WT. ( C ) Binding of OPG-WT and OPG-C3 to WT (CHO-K1) and HS-deficient (pgsD-677) CHO cells. Cell surface–bound OPG was quantified as described in Methods. ( D ) Association of endogenous OPG with the surface of WT and Ext1 -null (KO) primary osteoblasts. ( E ) Effect of heparitinase treatment (H’ase) on the association of endogenous OPG with the surface of WT primary osteoblasts. ( F ) Cell surface localization of endogenous OPG in primary human osteoblasts. Cultures of human osteoblasts were double labeled with anti-OPG monoclonal antibody and SiR actin. Control, staining without primary antibody; αOPG, staining with anti-OPG monoclonal antibody; αOPG + H’ase, staining with anti-OPG monoclonal antibody after heparitinase treatment of cells. Scale bar: 10 μm. ( G ) HS binding is necessary for OPG to efficiently inhibit osteoclastogenesis. Cocultures of osteoblasts (OB) and bone marrow macrophages (BM) were prepared in the combination of two OPG forms (OPG-WT [WT] and OPG-C3 [C3]) and osteoblasts of two genotypes (WT and KO), as indicated in the marker table on the right, and emergence of TRAP-positive osteoclasts was quantitated. OPGs were added at 0, 1, 10, 100, and 300 ng/ml, as indicated. Results are shown as the percentage of TRAP-positive cells relative to the total number of cells. Note that little osteoclastogenesis-inhibitory activity is detected in the OPG-WT/ Ext1 -null osteoblast combination (squares) and the OPG-C3/WT osteoblast combination (triangles) at 100 ng/ml, while the OPG-WT/WT osteoblast combination shows a significant inhibitory effects in the range of 1–10 ng/ml and almost complete inhibition at 100 ng/ml (circles). Data represent the mean ± SD (number of cultures tested = 5 in C , 4 in D , and 3 in E and F ). * P

    Article Snippet: Digestion of cell surface HS was performed by incubating live cells with 5 mIU/ml heparitinase (heparinase III) (MilliporeSigma, H8891) for 2 hours at 37°C.

    Techniques: Functional Assay, Generated, Affinity Chromatography, Binding Assay, Labeling, Staining, Marker, Activity Assay, Inhibition

    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

    Enzymatic cleavage of heparan sulfate prevents LTP but has no effect on single pulse-evoked synaptic responses in the area CA1 of hippocampal slices. A , Effect of HFS on the fEPSP slope in rat hippocampal slices (300 μm) preincubated with heparitinase–0.2% BSA (20 U/ml; volume, 500 μl; 3 hr; +24°C) (•) or 0.2% BSA only (○) (average ± SEM; n = 7 on both groups; p

    Journal: The Journal of Neuroscience

    Article Title: Reg1ulatory Role and Molecular Interactions of a Cell-Surface Heparan Sulfate Proteoglycan (N-syndecan) in Hippocampal Long-Term Potentiation

    doi: 10.1523/JNEUROSCI.19-04-01226.1999

    Figure Lengend Snippet: Enzymatic cleavage of heparan sulfate prevents LTP but has no effect on single pulse-evoked synaptic responses in the area CA1 of hippocampal slices. A , Effect of HFS on the fEPSP slope in rat hippocampal slices (300 μm) preincubated with heparitinase–0.2% BSA (20 U/ml; volume, 500 μl; 3 hr; +24°C) (•) or 0.2% BSA only (○) (average ± SEM; n = 7 on both groups; p

    Article Snippet: Aliquots of the fractions were digested with nitrous acid or heparitinase (10 U/ml, 15 hr) to remove carbohydrate side chains or incubated with 50 μ m herbimycin A (Calbiochem, La Jolla, CA).

    Techniques:

    Heparitinase treatment liberates FGF. Cartilage explants were incubated with buffer alone, heparitinase (1 mμ/ml), or chondroitinase ABC (5 mμ/ml). After 4 h each CM was removed (B CM, HT CM, and Ch CM, respectively) and assessed for their ability to activate ERK in isolated chondrocytes ( A Upper ). Rested cells (None), FGF alone or with heparitinase (FGF + HT) and heparitinase alone (HT) were assayed as controls. The blot was stripped and reprobed for total ERK ( A Lower ). CM from recut cartilage (RC CM) or from heparitinase-treated explants (HT CM) was preincubated with neutralizing FGF Abs (FGF) or with a nonimmune Ab (NI) for 2 h before stimulation of isolated chondrocytes ( B ). Cell lysates were immunoblotted for pERK ( B Upper ) and reprobed for total ERK ( B Lower ).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Basic FGF mediates an immediate response of articular cartilage to mechanical injury

    doi: 10.1073/pnas.122033199

    Figure Lengend Snippet: Heparitinase treatment liberates FGF. Cartilage explants were incubated with buffer alone, heparitinase (1 mμ/ml), or chondroitinase ABC (5 mμ/ml). After 4 h each CM was removed (B CM, HT CM, and Ch CM, respectively) and assessed for their ability to activate ERK in isolated chondrocytes ( A Upper ). Rested cells (None), FGF alone or with heparitinase (FGF + HT) and heparitinase alone (HT) were assayed as controls. The blot was stripped and reprobed for total ERK ( A Lower ). CM from recut cartilage (RC CM) or from heparitinase-treated explants (HT CM) was preincubated with neutralizing FGF Abs (FGF) or with a nonimmune Ab (NI) for 2 h before stimulation of isolated chondrocytes ( B ). Cell lysates were immunoblotted for pERK ( B Upper ) and reprobed for total ERK ( B Lower ).

    Article Snippet: Recombinant bFGF (146-aa length) was from PeproTech (London), and heparitinase was from Calbiochem.

    Techniques: Incubation, Isolation