Structured Review

AMS Biotechnology heparitinase
Chondroitin sulfate and Heparan sulfate are widely expressed in the developing zebrafish skeleton. A: Alcian blue- and alizarin red-stained skeletal preparations showing cartilage (blue) and mineralised tissue (red) at 4 and 8 dpf (lateral). B: Heparan sulfate labelling of the head with monoclonal antibody 3G10 at 6 dpf. C: Immunohistochemistry controls of 3G10 (labelling heparan sulphate) and CS56 (labelling native chondroitin sulphate), with and without chondroitinase <t>ABC/heparitinase</t> digestion as labelled at 4dpf. Heparitinase treatment is required to generate the epitope recognised by the 3G10 antibody, as such the heparitinase untreated fish show very limited immunoreactivity. CS56 antibody recognises a currently uncharacterised epitope present in native CS chains; treatment with chondroitinase ABC decreases immunoreactivity but doesn't completely prevent antibody binding. Ventral views with anterior to top. Inset in left-most panel with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. D: Chondroitin sulfate labelling of the head from 3–8 dpf with monoclonal antibody CS-56. E: Treatment of larvae with the GAG chain inhibitor PNPX leads to decreased GAG synthesis and decreased labelling with CS-56 in newly synthesised cartilage elements, demonstrating that CS-56 specifically labels CS chains. Images are all at 4dpf after treatment with PNPX (controls with DMSO) from 50 hpf. Left panels: Brightfield views of whole larvae to show that while morphology is relatively normal heart oedema is present. Second pair of panels: Flat-mounted cartilages of the ventral jaw stained with Alcian blue to reveal GAG content. Treatment with PNPX leads to significant reduction in cartilage GAG levels. Third pair of panels: Confocal stacks of the central jaw of zebrafish labelled with CS-56 antibody at 4dpf, treatment with PNPX leads to a significant reduction in cartilage labelling of CS-56 such that levels are comparable with the reduction in GAG synthesis observed by Alcian blue labelling. Right pair of panels: Tail of the zebrafish labelled with CS-56, comparable labelling of the notochord is seen following treatment with PNPX, likely because notochord synthesis of GAGs occurs between 24 and 48hpf prior to the onset of treatment with PNPX. Insets in images with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. mc, Meckel's cartilage; ch, ceratohyal; ba, branchial arches; op, operculum; ps, parasphenoid; oc, otic capsule; ot, otiliths; cl, cleithrum; 5ba, 5th branchial arch and teeth; nc, notochord; vb, developing vertebrae; ha, haemal arch; na, neural arch; sb, somite boundaries; +ve, positive; −ve, negative. Anterior is to left in all images. Scale bars = 100 μm in all panels.
Heparitinase, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/heparitinase/product/AMS Biotechnology
Average 91 stars, based on 11 article reviews
Price from $9.99 to $1999.99
heparitinase - by Bioz Stars, 2020-09
91/100 stars

Images

1) Product Images from "Expression of Glycosaminoglycan Epitopes During Zebrafish Skeletogenesis"

Article Title: Expression of Glycosaminoglycan Epitopes During Zebrafish Skeletogenesis

Journal: Developmental Dynamics

doi: 10.1002/dvdy.23970

Chondroitin sulfate and Heparan sulfate are widely expressed in the developing zebrafish skeleton. A: Alcian blue- and alizarin red-stained skeletal preparations showing cartilage (blue) and mineralised tissue (red) at 4 and 8 dpf (lateral). B: Heparan sulfate labelling of the head with monoclonal antibody 3G10 at 6 dpf. C: Immunohistochemistry controls of 3G10 (labelling heparan sulphate) and CS56 (labelling native chondroitin sulphate), with and without chondroitinase ABC/heparitinase digestion as labelled at 4dpf. Heparitinase treatment is required to generate the epitope recognised by the 3G10 antibody, as such the heparitinase untreated fish show very limited immunoreactivity. CS56 antibody recognises a currently uncharacterised epitope present in native CS chains; treatment with chondroitinase ABC decreases immunoreactivity but doesn't completely prevent antibody binding. Ventral views with anterior to top. Inset in left-most panel with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. D: Chondroitin sulfate labelling of the head from 3–8 dpf with monoclonal antibody CS-56. E: Treatment of larvae with the GAG chain inhibitor PNPX leads to decreased GAG synthesis and decreased labelling with CS-56 in newly synthesised cartilage elements, demonstrating that CS-56 specifically labels CS chains. Images are all at 4dpf after treatment with PNPX (controls with DMSO) from 50 hpf. Left panels: Brightfield views of whole larvae to show that while morphology is relatively normal heart oedema is present. Second pair of panels: Flat-mounted cartilages of the ventral jaw stained with Alcian blue to reveal GAG content. Treatment with PNPX leads to significant reduction in cartilage GAG levels. Third pair of panels: Confocal stacks of the central jaw of zebrafish labelled with CS-56 antibody at 4dpf, treatment with PNPX leads to a significant reduction in cartilage labelling of CS-56 such that levels are comparable with the reduction in GAG synthesis observed by Alcian blue labelling. Right pair of panels: Tail of the zebrafish labelled with CS-56, comparable labelling of the notochord is seen following treatment with PNPX, likely because notochord synthesis of GAGs occurs between 24 and 48hpf prior to the onset of treatment with PNPX. Insets in images with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. mc, Meckel's cartilage; ch, ceratohyal; ba, branchial arches; op, operculum; ps, parasphenoid; oc, otic capsule; ot, otiliths; cl, cleithrum; 5ba, 5th branchial arch and teeth; nc, notochord; vb, developing vertebrae; ha, haemal arch; na, neural arch; sb, somite boundaries; +ve, positive; −ve, negative. Anterior is to left in all images. Scale bars = 100 μm in all panels.
Figure Legend Snippet: Chondroitin sulfate and Heparan sulfate are widely expressed in the developing zebrafish skeleton. A: Alcian blue- and alizarin red-stained skeletal preparations showing cartilage (blue) and mineralised tissue (red) at 4 and 8 dpf (lateral). B: Heparan sulfate labelling of the head with monoclonal antibody 3G10 at 6 dpf. C: Immunohistochemistry controls of 3G10 (labelling heparan sulphate) and CS56 (labelling native chondroitin sulphate), with and without chondroitinase ABC/heparitinase digestion as labelled at 4dpf. Heparitinase treatment is required to generate the epitope recognised by the 3G10 antibody, as such the heparitinase untreated fish show very limited immunoreactivity. CS56 antibody recognises a currently uncharacterised epitope present in native CS chains; treatment with chondroitinase ABC decreases immunoreactivity but doesn't completely prevent antibody binding. Ventral views with anterior to top. Inset in left-most panel with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. D: Chondroitin sulfate labelling of the head from 3–8 dpf with monoclonal antibody CS-56. E: Treatment of larvae with the GAG chain inhibitor PNPX leads to decreased GAG synthesis and decreased labelling with CS-56 in newly synthesised cartilage elements, demonstrating that CS-56 specifically labels CS chains. Images are all at 4dpf after treatment with PNPX (controls with DMSO) from 50 hpf. Left panels: Brightfield views of whole larvae to show that while morphology is relatively normal heart oedema is present. Second pair of panels: Flat-mounted cartilages of the ventral jaw stained with Alcian blue to reveal GAG content. Treatment with PNPX leads to significant reduction in cartilage GAG levels. Third pair of panels: Confocal stacks of the central jaw of zebrafish labelled with CS-56 antibody at 4dpf, treatment with PNPX leads to a significant reduction in cartilage labelling of CS-56 such that levels are comparable with the reduction in GAG synthesis observed by Alcian blue labelling. Right pair of panels: Tail of the zebrafish labelled with CS-56, comparable labelling of the notochord is seen following treatment with PNPX, likely because notochord synthesis of GAGs occurs between 24 and 48hpf prior to the onset of treatment with PNPX. Insets in images with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. mc, Meckel's cartilage; ch, ceratohyal; ba, branchial arches; op, operculum; ps, parasphenoid; oc, otic capsule; ot, otiliths; cl, cleithrum; 5ba, 5th branchial arch and teeth; nc, notochord; vb, developing vertebrae; ha, haemal arch; na, neural arch; sb, somite boundaries; +ve, positive; −ve, negative. Anterior is to left in all images. Scale bars = 100 μm in all panels.

Techniques Used: Staining, Immunohistochemistry, Fluorescence In Situ Hybridization, Binding Assay

2) Product Images from "Heparanase activates the syndecan-syntenin-ALIX exosome pathway"

Article Title: Heparanase activates the syndecan-syntenin-ALIX exosome pathway

Journal: Cell Research

doi: 10.1038/cr.2015.29

Heparanase influences the biogenesis of vesicles of endosomal origin, enhancing intraluminal budding. (A) To investigate whether the extracellular vesicles affected by heparanase were of endosomal origin, RAB7 was knocked down ( RAB7 RNAi) in MCF-7 cells. Non-targeting RNAi (−) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of three independent experiments. (B) Quantification of the effect of RAB7 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF and CD63 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi with heparanase, dark gray bars; RAB7 knockdown, light gray bars; RAB7 knockdown with heparanase, white bars). Values are relative to the levels (intensities of the signals) measured in exosomes derived from cells treated with non-targeting RNAi in the absence of proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P
Figure Legend Snippet: Heparanase influences the biogenesis of vesicles of endosomal origin, enhancing intraluminal budding. (A) To investigate whether the extracellular vesicles affected by heparanase were of endosomal origin, RAB7 was knocked down ( RAB7 RNAi) in MCF-7 cells. Non-targeting RNAi (−) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of three independent experiments. (B) Quantification of the effect of RAB7 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF and CD63 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi with heparanase, dark gray bars; RAB7 knockdown, light gray bars; RAB7 knockdown with heparanase, white bars). Values are relative to the levels (intensities of the signals) measured in exosomes derived from cells treated with non-targeting RNAi in the absence of proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P

Techniques Used: Activity Assay, Migration, Molecular Weight, Western Blot, Derivative Assay

Heparanase acts through syntenin-1, ALIX and the syntenin-ALIX interaction to stimulate intraluminal budding. (A) The role of syntenin-1 and (B) the role of ALIX in the effect of heparanase on exosomes were investigated by the knockdown of syntenin-1 and ALIX (using Synt1 RNAi and ALIX RNAi, respectively) in MCF-7 cells. Non-targeting RNAi (NT) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both the experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of two independent experiments. Note that lysate and exosome samples derived from NT and KD cells, separated by a blank space in A , were run in the same gel, but not side by side, and that the band intensities in each row are directly comparable. (C) Confocal micrographs of MCF-7 cells co-transfected with wild-type mCherry-syntenin-1 (mCherry-Synt1 WT) or with mCherry-syntenin-1 harboring mutant LYP sequences and therefore defective in ALIX-binding (mCherry-Synt1-ΔALIX; red in merge), Cerulean-RAB5 Q79L (blue in merge) and syndecan-1 (green in merge). Intraluminal budding of wild-type and mutant mCherry-syntenin-1 and of syndecan-1 cytoplasmic domain was scored in the presence (Hep) or absence (no Hep) of heparanase. (D) Confocal micrographs of MCF-7 cells co-transfected with mCherry-syntenin-1 (red in merge) and Cerulean-RAB5 Q79L (blue in merge). In addition, the cells were transfected with RNAi targeting ALIX ( ALIX RNAi) or with non-targeting RNAi (NT RNAi). Intraluminal budding of mCherry-syntenin-1 was evaluated in the presence (Hep) or absence (no Hep) of heparanase. (E) Quantification of intraluminal budding of mCherry-syntenin-1 and syndecan-1 cytoplasmic domain, as in C and (F) quantitation of intraluminal budding of mCherry-syntenin-1, as in D , measuring the mean fluorescence intensity of mCherry-syntenin-1 and syndecan-1 in the lumen of RAB5 Q79L -positive endosomes (mean gray value per pixel). Bar heights represent mean values calculated from three independent experiments, scoring at least 30 cells per experiment. Individual data points are shown as white dots on top of the corresponding bars. ** P
Figure Legend Snippet: Heparanase acts through syntenin-1, ALIX and the syntenin-ALIX interaction to stimulate intraluminal budding. (A) The role of syntenin-1 and (B) the role of ALIX in the effect of heparanase on exosomes were investigated by the knockdown of syntenin-1 and ALIX (using Synt1 RNAi and ALIX RNAi, respectively) in MCF-7 cells. Non-targeting RNAi (NT) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both the experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of two independent experiments. Note that lysate and exosome samples derived from NT and KD cells, separated by a blank space in A , were run in the same gel, but not side by side, and that the band intensities in each row are directly comparable. (C) Confocal micrographs of MCF-7 cells co-transfected with wild-type mCherry-syntenin-1 (mCherry-Synt1 WT) or with mCherry-syntenin-1 harboring mutant LYP sequences and therefore defective in ALIX-binding (mCherry-Synt1-ΔALIX; red in merge), Cerulean-RAB5 Q79L (blue in merge) and syndecan-1 (green in merge). Intraluminal budding of wild-type and mutant mCherry-syntenin-1 and of syndecan-1 cytoplasmic domain was scored in the presence (Hep) or absence (no Hep) of heparanase. (D) Confocal micrographs of MCF-7 cells co-transfected with mCherry-syntenin-1 (red in merge) and Cerulean-RAB5 Q79L (blue in merge). In addition, the cells were transfected with RNAi targeting ALIX ( ALIX RNAi) or with non-targeting RNAi (NT RNAi). Intraluminal budding of mCherry-syntenin-1 was evaluated in the presence (Hep) or absence (no Hep) of heparanase. (E) Quantification of intraluminal budding of mCherry-syntenin-1 and syndecan-1 cytoplasmic domain, as in C and (F) quantitation of intraluminal budding of mCherry-syntenin-1, as in D , measuring the mean fluorescence intensity of mCherry-syntenin-1 and syndecan-1 in the lumen of RAB5 Q79L -positive endosomes (mean gray value per pixel). Bar heights represent mean values calculated from three independent experiments, scoring at least 30 cells per experiment. Individual data points are shown as white dots on top of the corresponding bars. ** P

Techniques Used: Activity Assay, Migration, Molecular Weight, Western Blot, Derivative Assay, Transfection, Mutagenesis, Binding Assay, Quantitation Assay, Fluorescence

Heparanase stimulates the production of syntenin-1-containing exosomes. (A) Exosome production was evaluated after overnight conditioning of MCF-7 cells with increasing concentrations of proheparanase (0.04-25 nM) and compared to that of cells not receiving proheparanase (0 nM). Exosomes were collected from equivalent amounts of culture medium, conditioned by equal numbers of cells, for equal lengths of time. For each condition both the lysate and exosomal fractions were analyzed by western blot, using cognate antibodies against heparanase, monitoring the conversion of proheparanase (Prohep) into mature heparanase (Hep) and against different exosomal markers: syntenin-1 (Synt1), syndecan-1 (SDC1), syndecan-4 (SDC4), CD63, flotillin-1 (Flo1), CD9 and CD81. Syndecan-1, which is a hybrid heparan sulfate (HS)/chondroitin sulfate proteoglycan, was analyzed using two different approaches. In one approach, the samples were digested with both heparitinase and chondroitinase ABC, removing all glycosaminoglycan chains and enabling visualization of the full-length syndecan core proteins (SDC1 FL) as sharp bands. In the other approach, the samples were digested with chondroitinase ABC only, leaving the HS on the syndecans (SDC1 with HS); comparison of 'SDC1 with HS' and 'SDC1 FL' yields information on the mass of HS on syndecans. Because of the heterogeneity in HS chain length, syndecan-1 with HS is smeared over a wide mass range in the absence of heparanase activity (and is therefore hardly visible in western blot, as illustrated by lane 1 of the lysates). With increasing heparanase activity, the HS chains on syndecan-1 are trimmed to shorter chains of more or less the same length, syndecan-1 with HS migrating as one or a few bands that are readily visualized in western blot (as illustrated by lane 6 of the lysates). Note that cell lysates contain mainly full-length syndecan core proteins; the opposite is true for exosomes, where hardly any full-length syndecan is detected and C-terminal fragments (CTFs) represent the dominant form. β-actin was used as a loading control for the lysates. Western blots are representative of five independent experiments. (B) Histogram representing the quantification of the exosomal levels of syntenin-1 (Synt1), syndecan-1 CTF (SDC1 CTF), CD63, syndecan-4 CTF (SDC4 CTF) and flotillin-1 (Flo1) in response to the addition of increasing concentrations (0 nM till 25 nM) of proheparanase. Values are relative to the exosomal levels measured in absence of exogenously added proheparanase. Bar heights represent mean values, calculated from five independent experiments. Individual data points are shown as white dots on top of the corresponding bars. * P
Figure Legend Snippet: Heparanase stimulates the production of syntenin-1-containing exosomes. (A) Exosome production was evaluated after overnight conditioning of MCF-7 cells with increasing concentrations of proheparanase (0.04-25 nM) and compared to that of cells not receiving proheparanase (0 nM). Exosomes were collected from equivalent amounts of culture medium, conditioned by equal numbers of cells, for equal lengths of time. For each condition both the lysate and exosomal fractions were analyzed by western blot, using cognate antibodies against heparanase, monitoring the conversion of proheparanase (Prohep) into mature heparanase (Hep) and against different exosomal markers: syntenin-1 (Synt1), syndecan-1 (SDC1), syndecan-4 (SDC4), CD63, flotillin-1 (Flo1), CD9 and CD81. Syndecan-1, which is a hybrid heparan sulfate (HS)/chondroitin sulfate proteoglycan, was analyzed using two different approaches. In one approach, the samples were digested with both heparitinase and chondroitinase ABC, removing all glycosaminoglycan chains and enabling visualization of the full-length syndecan core proteins (SDC1 FL) as sharp bands. In the other approach, the samples were digested with chondroitinase ABC only, leaving the HS on the syndecans (SDC1 with HS); comparison of 'SDC1 with HS' and 'SDC1 FL' yields information on the mass of HS on syndecans. Because of the heterogeneity in HS chain length, syndecan-1 with HS is smeared over a wide mass range in the absence of heparanase activity (and is therefore hardly visible in western blot, as illustrated by lane 1 of the lysates). With increasing heparanase activity, the HS chains on syndecan-1 are trimmed to shorter chains of more or less the same length, syndecan-1 with HS migrating as one or a few bands that are readily visualized in western blot (as illustrated by lane 6 of the lysates). Note that cell lysates contain mainly full-length syndecan core proteins; the opposite is true for exosomes, where hardly any full-length syndecan is detected and C-terminal fragments (CTFs) represent the dominant form. β-actin was used as a loading control for the lysates. Western blots are representative of five independent experiments. (B) Histogram representing the quantification of the exosomal levels of syntenin-1 (Synt1), syndecan-1 CTF (SDC1 CTF), CD63, syndecan-4 CTF (SDC4 CTF) and flotillin-1 (Flo1) in response to the addition of increasing concentrations (0 nM till 25 nM) of proheparanase. Values are relative to the exosomal levels measured in absence of exogenously added proheparanase. Bar heights represent mean values, calculated from five independent experiments. Individual data points are shown as white dots on top of the corresponding bars. * P

Techniques Used: Western Blot, Activity Assay

Related Articles

Western Blot:

Article Title: Heparanase activates the syndecan-syntenin-ALIX exosome pathway
Article Snippet: .. Western blot To allow detection of full-length syndecan core proteins, the samples were digested with (0.006 U/ml) heparitinase (Amsbio) and (0.166 U/ml) chondroitinase ABC (Amsbio) for 3 h at 37 °C in heparitinase buffer (0.1% Triton, 0.1 M NaCl, 1 mM CaCl2, 50 mM 6-aminohexanoic acid and 50 mM HEPES, at pH 7). .. Samples were fractionated in 4%-12% gradient gels (NuPAGE Novex Bis-Tris gels, Invitrogen), electro-transferred to Hybond-C extra 0.45-μm nitrocellulose membrane (GE Healthcare Life Sciences) and incubated with the indicated antibodies.

Incubation:

Article Title: A VP1 mutation acquired during an enterovirus 71 disseminated infection confers heparan sulfate binding ability and modulates ex vivo tropism
Article Snippet: .. Confluent Vero, Caco-2, RD or SH-SY5Y cells pre-plated in a 96 well plate, were washed twice with heparinase III digestion buffer (0.1 M sodium acetate pH 7.0, 1 mM calcium acetate and 0.2% BSA) and incubated for 1 h at 37°C with 3.5 mIU/ml of heparinase III (AMS.HEP_ENZ III_S, Amsbio, Switzerland) (50 μl/well). .. Control samples were incubated with digestion buffer alone.

Article Title: A VP1 mutation acquired during an enterovirus 71 disseminated infection confers heparan sulfate binding ability and modulates ex vivo tropism
Article Snippet: .. Heparinase assay Confluent Vero, Caco-2, RD or SH-SY5Y cells pre-plated in a 96 well plate, were washed twice with heparinase III digestion buffer (0.1 M sodium acetate pH 7.0, 1 mM calcium acetate and 0.2% BSA) and incubated for 1 h at 37°C with 3.5 mIU/ml of heparinase III (AMS.HEP_ENZ III_S, Amsbio, Switzerland) (50 μl/well). .. Control samples were incubated with digestion buffer alone.

Affinity Magnetic Separation:

Article Title: A VP1 mutation acquired during an enterovirus 71 disseminated infection confers heparan sulfate binding ability and modulates ex vivo tropism
Article Snippet: .. Confluent Vero, Caco-2, RD or SH-SY5Y cells pre-plated in a 96 well plate, were washed twice with heparinase III digestion buffer (0.1 M sodium acetate pH 7.0, 1 mM calcium acetate and 0.2% BSA) and incubated for 1 h at 37°C with 3.5 mIU/ml of heparinase III (AMS.HEP_ENZ III_S, Amsbio, Switzerland) (50 μl/well). .. Control samples were incubated with digestion buffer alone.

Article Title: A VP1 mutation acquired during an enterovirus 71 disseminated infection confers heparan sulfate binding ability and modulates ex vivo tropism
Article Snippet: .. Heparinase assay Confluent Vero, Caco-2, RD or SH-SY5Y cells pre-plated in a 96 well plate, were washed twice with heparinase III digestion buffer (0.1 M sodium acetate pH 7.0, 1 mM calcium acetate and 0.2% BSA) and incubated for 1 h at 37°C with 3.5 mIU/ml of heparinase III (AMS.HEP_ENZ III_S, Amsbio, Switzerland) (50 μl/well). .. Control samples were incubated with digestion buffer alone.

SDS Page:

Article Title: Syndecan1 is a critical mediator of macropinocytosis in pancreatic cancer
Article Snippet: .. Soluble material was resuspended in lysase buffer containing 0.0001 units heparitinase (Amsbio) and 0.005 units chondroitin ABC lysase (Sigma) and were proceeded to be resolved by 4–12% SDS-PAGE, transferred to Immobilon-P PVDF, fixed in 0.05% glutaraldehyde (Sigma). .. Primary antibodies used for IHC and Western blot analysis are listed in the Extended Experimental Procedures.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    AMS Biotechnology heparinase iii
    Heparan sulfates are crucial for SARS-CoV-2 binding and infection. ( A-B ) Huh 7.5 cells were preincubated with neutralizing antibody toACE2 and SARS-CoV-2 pseudovirus was pre-incubated with mAb COVA1-18, COVA1-21 and COVA2-15 or UF heparin (250IU). Cells were infected with SARS-CoV-2 pseudovirus and binding was determined by ELISA. ( C ) Heparan sulfates were removed from Huh 7.5 cells by <t>heparinase</t> treatment and SARS-CoV-2 pseudovirus binding was determined. Error bars are the mean ± SEM from <t>three</t> independent experiments. ( D ) Huh 7.5 cells were infected with SARS-CoV-2 pseudovirus and infection was measured after 5 days of culture by luciferase assay. Virus was pretreated with heparin (2501U). Data show the mean values and error bars are the SEM. Statistical analysis was performed using ( A ) ordinary one-way ANOVAwith Tukey multiple-comparison test. ***p= 0.0006, ***p= 0.0005, **p= 0.0018, **p= 0.0036 (n = 3), ( B ) **p= 0.0010, *p= 0.0255 (n=4), ( C ) two-tailed, unpaired Student’s t-test with Welch’s correction **p= 0.0012 (n = 3), ( D ) ordinary one-way ANOVAwith Tukey multiple-comparison test. **p= 0.0085 (n=6 measured in triplicate). RLU: relative light units.
    Heparinase Iii, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparinase iii/product/AMS Biotechnology
    Average 92 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    heparinase iii - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    94
    AMS Biotechnology miliunits heparinase iii
    Heparan sulfates are crucial for SARS-CoV-2 binding and infection. ( A-B ) Huh 7.5 cells were preincubated with neutralizing antibody toACE2 and SARS-CoV-2 pseudovirus was pre-incubated with mAb COVA1-18, COVA1-21 and COVA2-15 or UF heparin (250IU). Cells were infected with SARS-CoV-2 pseudovirus and binding was determined by ELISA. ( C ) Heparan sulfates were removed from Huh 7.5 cells by <t>heparinase</t> treatment and SARS-CoV-2 pseudovirus binding was determined. Error bars are the mean ± SEM from <t>three</t> independent experiments. ( D ) Huh 7.5 cells were infected with SARS-CoV-2 pseudovirus and infection was measured after 5 days of culture by luciferase assay. Virus was pretreated with heparin (2501U). Data show the mean values and error bars are the SEM. Statistical analysis was performed using ( A ) ordinary one-way ANOVAwith Tukey multiple-comparison test. ***p= 0.0006, ***p= 0.0005, **p= 0.0018, **p= 0.0036 (n = 3), ( B ) **p= 0.0010, *p= 0.0255 (n=4), ( C ) two-tailed, unpaired Student’s t-test with Welch’s correction **p= 0.0012 (n = 3), ( D ) ordinary one-way ANOVAwith Tukey multiple-comparison test. **p= 0.0085 (n=6 measured in triplicate). RLU: relative light units.
    Miliunits Heparinase Iii, supplied by AMS Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/miliunits heparinase iii/product/AMS Biotechnology
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    miliunits heparinase iii - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    Image Search Results


    Heparan sulfates are crucial for SARS-CoV-2 binding and infection. ( A-B ) Huh 7.5 cells were preincubated with neutralizing antibody toACE2 and SARS-CoV-2 pseudovirus was pre-incubated with mAb COVA1-18, COVA1-21 and COVA2-15 or UF heparin (250IU). Cells were infected with SARS-CoV-2 pseudovirus and binding was determined by ELISA. ( C ) Heparan sulfates were removed from Huh 7.5 cells by heparinase treatment and SARS-CoV-2 pseudovirus binding was determined. Error bars are the mean ± SEM from three independent experiments. ( D ) Huh 7.5 cells were infected with SARS-CoV-2 pseudovirus and infection was measured after 5 days of culture by luciferase assay. Virus was pretreated with heparin (2501U). Data show the mean values and error bars are the SEM. Statistical analysis was performed using ( A ) ordinary one-way ANOVAwith Tukey multiple-comparison test. ***p= 0.0006, ***p= 0.0005, **p= 0.0018, **p= 0.0036 (n = 3), ( B ) **p= 0.0010, *p= 0.0255 (n=4), ( C ) two-tailed, unpaired Student’s t-test with Welch’s correction **p= 0.0012 (n = 3), ( D ) ordinary one-way ANOVAwith Tukey multiple-comparison test. **p= 0.0085 (n=6 measured in triplicate). RLU: relative light units.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Infection and Transmission Depends on Heparan Sulfates and Is Blocked by Low Molecular Weight Heparins

    doi: 10.1101/2020.08.18.255810

    Figure Lengend Snippet: Heparan sulfates are crucial for SARS-CoV-2 binding and infection. ( A-B ) Huh 7.5 cells were preincubated with neutralizing antibody toACE2 and SARS-CoV-2 pseudovirus was pre-incubated with mAb COVA1-18, COVA1-21 and COVA2-15 or UF heparin (250IU). Cells were infected with SARS-CoV-2 pseudovirus and binding was determined by ELISA. ( C ) Heparan sulfates were removed from Huh 7.5 cells by heparinase treatment and SARS-CoV-2 pseudovirus binding was determined. Error bars are the mean ± SEM from three independent experiments. ( D ) Huh 7.5 cells were infected with SARS-CoV-2 pseudovirus and infection was measured after 5 days of culture by luciferase assay. Virus was pretreated with heparin (2501U). Data show the mean values and error bars are the SEM. Statistical analysis was performed using ( A ) ordinary one-way ANOVAwith Tukey multiple-comparison test. ***p= 0.0006, ***p= 0.0005, **p= 0.0018, **p= 0.0036 (n = 3), ( B ) **p= 0.0010, *p= 0.0255 (n=4), ( C ) two-tailed, unpaired Student’s t-test with Welch’s correction **p= 0.0012 (n = 3), ( D ) ordinary one-way ANOVAwith Tukey multiple-comparison test. **p= 0.0085 (n=6 measured in triplicate). RLU: relative light units.

    Article Snippet: Heparinase III from Flavobacterium heparium, EC 4.2.2.8, Batch 010, (Amsbio).

    Techniques: Binding Assay, Infection, Incubation, Enzyme-linked Immunosorbent Assay, Luciferase, Two Tailed Test

    ( A ) ACE2 cell surface expression on Namatwa cell line, DCs, LCs and Huh 7.5. Representative data for an experiment repeated more than three times with similar results. ( B ) Huh7.5 were left untreated or treated with heparinase for 1 hand heparan sulfate or digested heparan sulfate expression was determined by flow cytometry. One representative donor out of 3 is depicted. ( C ) Cell surface expression of ACE2 on 293T and Caco2 cell lines was determined by real-time PCR. ( D ) LC were stained with antibodies against CD207 and CD1 a and analysed by flow cytometry. The histogram shows the cell surface expression of the receptor. Representative data for an experiment repeated more than three times with similar results. Data show the mean values and error bars are the SEM. Statistical analysis was performed using ( A ) ordinary one-way ANOVA with Tukey’s multiple-comparison test. ****p

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Infection and Transmission Depends on Heparan Sulfates and Is Blocked by Low Molecular Weight Heparins

    doi: 10.1101/2020.08.18.255810

    Figure Lengend Snippet: ( A ) ACE2 cell surface expression on Namatwa cell line, DCs, LCs and Huh 7.5. Representative data for an experiment repeated more than three times with similar results. ( B ) Huh7.5 were left untreated or treated with heparinase for 1 hand heparan sulfate or digested heparan sulfate expression was determined by flow cytometry. One representative donor out of 3 is depicted. ( C ) Cell surface expression of ACE2 on 293T and Caco2 cell lines was determined by real-time PCR. ( D ) LC were stained with antibodies against CD207 and CD1 a and analysed by flow cytometry. The histogram shows the cell surface expression of the receptor. Representative data for an experiment repeated more than three times with similar results. Data show the mean values and error bars are the SEM. Statistical analysis was performed using ( A ) ordinary one-way ANOVA with Tukey’s multiple-comparison test. ****p

    Article Snippet: Heparinase III from Flavobacterium heparium, EC 4.2.2.8, Batch 010, (Amsbio).

    Techniques: Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction, Staining

    Chondroitin sulfate and Heparan sulfate are widely expressed in the developing zebrafish skeleton. A: Alcian blue- and alizarin red-stained skeletal preparations showing cartilage (blue) and mineralised tissue (red) at 4 and 8 dpf (lateral). B: Heparan sulfate labelling of the head with monoclonal antibody 3G10 at 6 dpf. C: Immunohistochemistry controls of 3G10 (labelling heparan sulphate) and CS56 (labelling native chondroitin sulphate), with and without chondroitinase ABC/heparitinase digestion as labelled at 4dpf. Heparitinase treatment is required to generate the epitope recognised by the 3G10 antibody, as such the heparitinase untreated fish show very limited immunoreactivity. CS56 antibody recognises a currently uncharacterised epitope present in native CS chains; treatment with chondroitinase ABC decreases immunoreactivity but doesn't completely prevent antibody binding. Ventral views with anterior to top. Inset in left-most panel with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. D: Chondroitin sulfate labelling of the head from 3–8 dpf with monoclonal antibody CS-56. E: Treatment of larvae with the GAG chain inhibitor PNPX leads to decreased GAG synthesis and decreased labelling with CS-56 in newly synthesised cartilage elements, demonstrating that CS-56 specifically labels CS chains. Images are all at 4dpf after treatment with PNPX (controls with DMSO) from 50 hpf. Left panels: Brightfield views of whole larvae to show that while morphology is relatively normal heart oedema is present. Second pair of panels: Flat-mounted cartilages of the ventral jaw stained with Alcian blue to reveal GAG content. Treatment with PNPX leads to significant reduction in cartilage GAG levels. Third pair of panels: Confocal stacks of the central jaw of zebrafish labelled with CS-56 antibody at 4dpf, treatment with PNPX leads to a significant reduction in cartilage labelling of CS-56 such that levels are comparable with the reduction in GAG synthesis observed by Alcian blue labelling. Right pair of panels: Tail of the zebrafish labelled with CS-56, comparable labelling of the notochord is seen following treatment with PNPX, likely because notochord synthesis of GAGs occurs between 24 and 48hpf prior to the onset of treatment with PNPX. Insets in images with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. mc, Meckel's cartilage; ch, ceratohyal; ba, branchial arches; op, operculum; ps, parasphenoid; oc, otic capsule; ot, otiliths; cl, cleithrum; 5ba, 5th branchial arch and teeth; nc, notochord; vb, developing vertebrae; ha, haemal arch; na, neural arch; sb, somite boundaries; +ve, positive; −ve, negative. Anterior is to left in all images. Scale bars = 100 μm in all panels.

    Journal: Developmental Dynamics

    Article Title: Expression of Glycosaminoglycan Epitopes During Zebrafish Skeletogenesis

    doi: 10.1002/dvdy.23970

    Figure Lengend Snippet: Chondroitin sulfate and Heparan sulfate are widely expressed in the developing zebrafish skeleton. A: Alcian blue- and alizarin red-stained skeletal preparations showing cartilage (blue) and mineralised tissue (red) at 4 and 8 dpf (lateral). B: Heparan sulfate labelling of the head with monoclonal antibody 3G10 at 6 dpf. C: Immunohistochemistry controls of 3G10 (labelling heparan sulphate) and CS56 (labelling native chondroitin sulphate), with and without chondroitinase ABC/heparitinase digestion as labelled at 4dpf. Heparitinase treatment is required to generate the epitope recognised by the 3G10 antibody, as such the heparitinase untreated fish show very limited immunoreactivity. CS56 antibody recognises a currently uncharacterised epitope present in native CS chains; treatment with chondroitinase ABC decreases immunoreactivity but doesn't completely prevent antibody binding. Ventral views with anterior to top. Inset in left-most panel with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. D: Chondroitin sulfate labelling of the head from 3–8 dpf with monoclonal antibody CS-56. E: Treatment of larvae with the GAG chain inhibitor PNPX leads to decreased GAG synthesis and decreased labelling with CS-56 in newly synthesised cartilage elements, demonstrating that CS-56 specifically labels CS chains. Images are all at 4dpf after treatment with PNPX (controls with DMSO) from 50 hpf. Left panels: Brightfield views of whole larvae to show that while morphology is relatively normal heart oedema is present. Second pair of panels: Flat-mounted cartilages of the ventral jaw stained with Alcian blue to reveal GAG content. Treatment with PNPX leads to significant reduction in cartilage GAG levels. Third pair of panels: Confocal stacks of the central jaw of zebrafish labelled with CS-56 antibody at 4dpf, treatment with PNPX leads to a significant reduction in cartilage labelling of CS-56 such that levels are comparable with the reduction in GAG synthesis observed by Alcian blue labelling. Right pair of panels: Tail of the zebrafish labelled with CS-56, comparable labelling of the notochord is seen following treatment with PNPX, likely because notochord synthesis of GAGs occurs between 24 and 48hpf prior to the onset of treatment with PNPX. Insets in images with low levels/no labelling of antibodies show DAPI stained or brightfield images for orientation. mc, Meckel's cartilage; ch, ceratohyal; ba, branchial arches; op, operculum; ps, parasphenoid; oc, otic capsule; ot, otiliths; cl, cleithrum; 5ba, 5th branchial arch and teeth; nc, notochord; vb, developing vertebrae; ha, haemal arch; na, neural arch; sb, somite boundaries; +ve, positive; −ve, negative. Anterior is to left in all images. Scale bars = 100 μm in all panels.

    Article Snippet: To generate the reactive HS neoepitope recognised by mAb 3G10, larvae were pre-digested with 5 mU/ml heparitinase (Amsbio, Lake Forest, CA) in 50 mM sodium acetate buffer (pH 7.0) containing 5 mM CaCl2 for 1 hr at 37°C, and re-fixed in 4% PFA.

    Techniques: Staining, Immunohistochemistry, Fluorescence In Situ Hybridization, Binding Assay

    Organization and Differentiation of Endodermal Cells is Dependent on HS Proteoglycans on Decellularized Lung Scaffolds (A) Acellular scaffolds were recellularized after heparitinase I or chondroitinase ABC treatment. H E staining of day 21 seeded scaffold cultures show limited organization and differentiation in the heparitinase I-treated group, while chondroitinase ABC-treated cultures resemble control groups. Scale bar represents 50 μm. (B) Scanning EM analysis of cultures show a lack of epithelial morphology and tight junction coupling of seeded cells in heparitinase I-treated scaffolds, where cells appear rounded with no resemblance to a lung phenotype. Scale bar represents 10 μm. (C) Proteome profiler antibody array detects 31 proteins from the array profile that are remaining on lung scaffolds (black). Comparison of the protein profile from decellularized scaffolds treated with or without heparitinase I revealed several HS-bound proteins that are removed from scaffolds and found in the wash supernatant after enzyme treatment (red rectangles): CXCL12, serpinE1, PDGF-AB, HGF, MMP8, FGF2, proliferin, IL10, and CCL3. Data presented are average of two arrays from separate experiments. See also Figure S5 .

    Journal: Stem Cell Reports

    Article Title: Acellular Lung Scaffolds Direct Differentiation of Endoderm to Functional Airway Epithelial Cells: Requirement of Matrix-Bound HS Proteoglycans

    doi: 10.1016/j.stemcr.2015.01.004

    Figure Lengend Snippet: Organization and Differentiation of Endodermal Cells is Dependent on HS Proteoglycans on Decellularized Lung Scaffolds (A) Acellular scaffolds were recellularized after heparitinase I or chondroitinase ABC treatment. H E staining of day 21 seeded scaffold cultures show limited organization and differentiation in the heparitinase I-treated group, while chondroitinase ABC-treated cultures resemble control groups. Scale bar represents 50 μm. (B) Scanning EM analysis of cultures show a lack of epithelial morphology and tight junction coupling of seeded cells in heparitinase I-treated scaffolds, where cells appear rounded with no resemblance to a lung phenotype. Scale bar represents 10 μm. (C) Proteome profiler antibody array detects 31 proteins from the array profile that are remaining on lung scaffolds (black). Comparison of the protein profile from decellularized scaffolds treated with or without heparitinase I revealed several HS-bound proteins that are removed from scaffolds and found in the wash supernatant after enzyme treatment (red rectangles): CXCL12, serpinE1, PDGF-AB, HGF, MMP8, FGF2, proliferin, IL10, and CCL3. Data presented are average of two arrays from separate experiments. See also Figure S5 .

    Article Snippet: Scaffold Enzymatic Treatment HS or CS proteoglycans were cleaved from decellularized lung scaffolds using treatment with heparitinase I (Amsbio #100704) or chondroitinase ABC (Amsbio #100330-1A), respectively.

    Techniques: Staining, Ab Array

    Heparan sulfates are crucial for SARS-CoV-2 binding and infection. ( A-B ) Huh 7.5 cells were preincubated with neutralizing antibody toACE2 and SARS-CoV-2 pseudovirus was pre-incubated with mAb COVA1-18, COVA1-21 and COVA2-15 or UF heparin (250IU). Cells were infected with SARS-CoV-2 pseudovirus and binding was determined by ELISA. ( C ) Heparan sulfates were removed from Huh 7.5 cells by heparinase treatment and SARS-CoV-2 pseudovirus binding was determined. Error bars are the mean ± SEM from three independent experiments. ( D ) Huh 7.5 cells were infected with SARS-CoV-2 pseudovirus and infection was measured after 5 days of culture by luciferase assay. Virus was pretreated with heparin (2501U). Data show the mean values and error bars are the SEM. Statistical analysis was performed using ( A ) ordinary one-way ANOVAwith Tukey multiple-comparison test. ***p= 0.0006, ***p= 0.0005, **p= 0.0018, **p= 0.0036 (n = 3), ( B ) **p= 0.0010, *p= 0.0255 (n=4), ( C ) two-tailed, unpaired Student’s t-test with Welch’s correction **p= 0.0012 (n = 3), ( D ) ordinary one-way ANOVAwith Tukey multiple-comparison test. **p= 0.0085 (n=6 measured in triplicate). RLU: relative light units.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Infection and Transmission Depends on Heparan Sulfates and Is Blocked by Low Molecular Weight Heparins

    doi: 10.1101/2020.08.18.255810

    Figure Lengend Snippet: Heparan sulfates are crucial for SARS-CoV-2 binding and infection. ( A-B ) Huh 7.5 cells were preincubated with neutralizing antibody toACE2 and SARS-CoV-2 pseudovirus was pre-incubated with mAb COVA1-18, COVA1-21 and COVA2-15 or UF heparin (250IU). Cells were infected with SARS-CoV-2 pseudovirus and binding was determined by ELISA. ( C ) Heparan sulfates were removed from Huh 7.5 cells by heparinase treatment and SARS-CoV-2 pseudovirus binding was determined. Error bars are the mean ± SEM from three independent experiments. ( D ) Huh 7.5 cells were infected with SARS-CoV-2 pseudovirus and infection was measured after 5 days of culture by luciferase assay. Virus was pretreated with heparin (2501U). Data show the mean values and error bars are the SEM. Statistical analysis was performed using ( A ) ordinary one-way ANOVAwith Tukey multiple-comparison test. ***p= 0.0006, ***p= 0.0005, **p= 0.0018, **p= 0.0036 (n = 3), ( B ) **p= 0.0010, *p= 0.0255 (n=4), ( C ) two-tailed, unpaired Student’s t-test with Welch’s correction **p= 0.0012 (n = 3), ( D ) ordinary one-way ANOVAwith Tukey multiple-comparison test. **p= 0.0085 (n=6 measured in triplicate). RLU: relative light units.

    Article Snippet: Biosynthesis inhibition and enzymatic treatmentHuH7.5 cells were treated in D-PBS/0.25% BSA with 46 miliunits heparinase III (Amsbio) for 1 hour at 37°C, washed and used in subsequent experiments.

    Techniques: Binding Assay, Infection, Incubation, Enzyme-linked Immunosorbent Assay, Luciferase, Two Tailed Test

    ( A ) ACE2 cell surface expression on Namatwa cell line, DCs, LCs and Huh 7.5. Representative data for an experiment repeated more than three times with similar results. ( B ) Huh7.5 were left untreated or treated with heparinase for 1 hand heparan sulfate or digested heparan sulfate expression was determined by flow cytometry. One representative donor out of 3 is depicted. ( C ) Cell surface expression of ACE2 on 293T and Caco2 cell lines was determined by real-time PCR. ( D ) LC were stained with antibodies against CD207 and CD1 a and analysed by flow cytometry. The histogram shows the cell surface expression of the receptor. Representative data for an experiment repeated more than three times with similar results. Data show the mean values and error bars are the SEM. Statistical analysis was performed using ( A ) ordinary one-way ANOVA with Tukey’s multiple-comparison test. ****p

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Infection and Transmission Depends on Heparan Sulfates and Is Blocked by Low Molecular Weight Heparins

    doi: 10.1101/2020.08.18.255810

    Figure Lengend Snippet: ( A ) ACE2 cell surface expression on Namatwa cell line, DCs, LCs and Huh 7.5. Representative data for an experiment repeated more than three times with similar results. ( B ) Huh7.5 were left untreated or treated with heparinase for 1 hand heparan sulfate or digested heparan sulfate expression was determined by flow cytometry. One representative donor out of 3 is depicted. ( C ) Cell surface expression of ACE2 on 293T and Caco2 cell lines was determined by real-time PCR. ( D ) LC were stained with antibodies against CD207 and CD1 a and analysed by flow cytometry. The histogram shows the cell surface expression of the receptor. Representative data for an experiment repeated more than three times with similar results. Data show the mean values and error bars are the SEM. Statistical analysis was performed using ( A ) ordinary one-way ANOVA with Tukey’s multiple-comparison test. ****p

    Article Snippet: Biosynthesis inhibition and enzymatic treatmentHuH7.5 cells were treated in D-PBS/0.25% BSA with 46 miliunits heparinase III (Amsbio) for 1 hour at 37°C, washed and used in subsequent experiments.

    Techniques: Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction, Staining