bacteroides heparinase iii  (New England Biolabs)


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    Name:
    Bacteroides Heparinase I
    Description:
    Bacteroides Heparinase I 600 units
    Catalog Number:
    p0735l
    Price:
    312
    Size:
    600 units
    Category:
    Glycosidases
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    Structured Review

    New England Biolabs bacteroides heparinase iii
    Bacteroides Heparinase I
    Bacteroides Heparinase I 600 units
    https://www.bioz.com/result/bacteroides heparinase iii/product/New England Biolabs
    Average 95 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    bacteroides heparinase iii - by Bioz Stars, 2020-08
    95/100 stars

    Images

    1) Product Images from "A Naturally Occurring Polymorphism in the HIV-1 Tat Basic Domain Inhibits Uptake by Bystander Cells and Leads to Reduced Neuroinflammation"

    Article Title: A Naturally Occurring Polymorphism in the HIV-1 Tat Basic Domain Inhibits Uptake by Bystander Cells and Leads to Reduced Neuroinflammation

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39531-5

    Flow cytometry studies indicate a higher uptake of CPP-R57 over CPP-S57 under a variety of conditions. Various cell types were incubated with fluorescently labeled CPP peptides and the uptake measured by flow cytometry. ( a ) 293T cells exposed to 2 μM of a control peptide, CPP-R57 or CPP-S57 peptides followed by analysis by flow cytometry. Proportional MFI values between experiments (n = 4) were then averaged and plotted on a histogram. Raw MFI values for each sample across different experiments spanned the following ranges: control (1.8–5.5), CPP-R57 (41.9–301.0) and CPP-S57 (11.6–99.0). ( b ) 293T cells and THP-1 cells exposed to increasing concentrations of CPPs as indicated to study dose response. ( c ) 293T cells exposed to 2 μM of CPPs at either 37 °C or 4 °C. Data was combined between different experiments (n = 4), and raw MFI values spanned the following ranges: control (2.8–5.3), CPP-R57 (58.8–266.0) and CPP-S57 (20.0–97.3). ( d ) 293T cells were exposed to CPPs at the indicated concentrations of heparin. ( e ) 293T cells treated for 2 h with the indicated concentrations of Heparinase I enzyme (with Heparinase III enzyme included at 1/17 the concentration of Heparinase I), then exposed to 2 μM of CPP. Data was combined between different experiments (n = 2), and raw MFI values spanned the following ranges: control 0 U (2.1–2.3), CPP-R57 0 U (114.0–145.0), CPP-S57 0 U (18.3–31.9), CPP-R57 1.9 U (64.6–75.1), CPP-S57 1.9 U (7.4–10.9), CPP-R57 3.8 U (46.3–49.9), CPP-S57 3.8 U (6.1–6.8), CPP-R57 7.5 U (34.1–35.4), CPP-S57 7.5 U (5.0–5.2), CPP-R57 15 U (27.8–35.2), and CPP-S57 15 U (4.0–5.0) (***p
    Figure Legend Snippet: Flow cytometry studies indicate a higher uptake of CPP-R57 over CPP-S57 under a variety of conditions. Various cell types were incubated with fluorescently labeled CPP peptides and the uptake measured by flow cytometry. ( a ) 293T cells exposed to 2 μM of a control peptide, CPP-R57 or CPP-S57 peptides followed by analysis by flow cytometry. Proportional MFI values between experiments (n = 4) were then averaged and plotted on a histogram. Raw MFI values for each sample across different experiments spanned the following ranges: control (1.8–5.5), CPP-R57 (41.9–301.0) and CPP-S57 (11.6–99.0). ( b ) 293T cells and THP-1 cells exposed to increasing concentrations of CPPs as indicated to study dose response. ( c ) 293T cells exposed to 2 μM of CPPs at either 37 °C or 4 °C. Data was combined between different experiments (n = 4), and raw MFI values spanned the following ranges: control (2.8–5.3), CPP-R57 (58.8–266.0) and CPP-S57 (20.0–97.3). ( d ) 293T cells were exposed to CPPs at the indicated concentrations of heparin. ( e ) 293T cells treated for 2 h with the indicated concentrations of Heparinase I enzyme (with Heparinase III enzyme included at 1/17 the concentration of Heparinase I), then exposed to 2 μM of CPP. Data was combined between different experiments (n = 2), and raw MFI values spanned the following ranges: control 0 U (2.1–2.3), CPP-R57 0 U (114.0–145.0), CPP-S57 0 U (18.3–31.9), CPP-R57 1.9 U (64.6–75.1), CPP-S57 1.9 U (7.4–10.9), CPP-R57 3.8 U (46.3–49.9), CPP-S57 3.8 U (6.1–6.8), CPP-R57 7.5 U (34.1–35.4), CPP-S57 7.5 U (5.0–5.2), CPP-R57 15 U (27.8–35.2), and CPP-S57 15 U (4.0–5.0) (***p

    Techniques Used: Flow Cytometry, Cytometry, Conditioned Place Preference, Incubation, Labeling, Concentration Assay

    2) Product Images from "Heparin affinity purification of extracellular vesicles"

    Article Title: Heparin affinity purification of extracellular vesicles

    Journal: Scientific Reports

    doi: 10.1038/srep10266

    Extracellular vesicles are efficiently isolated and purified using heparin-coated agarose beads. (a) Heparin coated agarose beads are incubated with EVs released from a variety of cells lines, (i), to yield an EV/heparin complex, (ii). Free floating proteins and nucleic acids are washed away with PBS, (iii). Beads are the incubated overnight with 2.15 M NaCl and the EVs are released and collected by spinning down the beads and collecting the supernatant (iv). Collected EVs are used as a source of RNA (biomarker) or used in biological assays (v ). (b) Nanoparticle tracking analysis (NTA) counts of heparin-purified human 293T-derived EVs eluted with 2.15 M NaCl overnight at 4 °C following 3 wash steps. ( c ) To show specific heparin affinity we incubated heparin beads overnight with EVs, then rinsed beads 3 times with PBS and treated with Bacteroides Heparinase I or incubation buffer without heparinase and fractions were analyzed by NTA. ( d ) EVs were mixed with heparin beads and one round of purification was performed. The unbound and eluted fractions from round one were separately incubated with a fresh batch of heparin beads and round 2 purification performed on these samples. NTA was performed on each fraction of round 2 purification.
    Figure Legend Snippet: Extracellular vesicles are efficiently isolated and purified using heparin-coated agarose beads. (a) Heparin coated agarose beads are incubated with EVs released from a variety of cells lines, (i), to yield an EV/heparin complex, (ii). Free floating proteins and nucleic acids are washed away with PBS, (iii). Beads are the incubated overnight with 2.15 M NaCl and the EVs are released and collected by spinning down the beads and collecting the supernatant (iv). Collected EVs are used as a source of RNA (biomarker) or used in biological assays (v ). (b) Nanoparticle tracking analysis (NTA) counts of heparin-purified human 293T-derived EVs eluted with 2.15 M NaCl overnight at 4 °C following 3 wash steps. ( c ) To show specific heparin affinity we incubated heparin beads overnight with EVs, then rinsed beads 3 times with PBS and treated with Bacteroides Heparinase I or incubation buffer without heparinase and fractions were analyzed by NTA. ( d ) EVs were mixed with heparin beads and one round of purification was performed. The unbound and eluted fractions from round one were separately incubated with a fresh batch of heparin beads and round 2 purification performed on these samples. NTA was performed on each fraction of round 2 purification.

    Techniques Used: Isolation, Purification, Incubation, Biomarker Assay, Derivative Assay

    3) Product Images from "Epigallocatechin-3-gallate remodels apolipoprotein A-I amyloid fibrils into soluble oligomers in the presence of heparin"

    Article Title: Epigallocatechin-3-gallate remodels apolipoprotein A-I amyloid fibrils into soluble oligomers in the presence of heparin

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.002038

    ApoA-I fibril formation in the absence or presence of heparin. A,  negative stain TEM images of apoA-I aggregates formed at pH 4 in the absence of heparin.  B , TEM images of apoA-I fibrils formed in the presence of 14–15 kDa of heparin (2-fold  m  excess).  C,  distribution and means of fibril widths measured from TEM images of apoA-I ± heparin.  D,  determination of heparin association with apoA-I fibrils.  Left , time course of uronic acid generation resulting from heparin cleavage by heparinase I.  Right , calculation of heparin remaining in solution following sedimentation with the protein at different molar ratios. Δ A 232  was measured as the end point  A 232  value minus the initial  A 232 . All initial solutions contained 1 mg/ml heparin (∼72 μ m ) before the addition of apoA-I and sedimentation of the aggregates formed. The concentrations shown represent the amount of heparin remaining in solution after removal of the insoluble material.  E,  fluorescence lifetime image (obtained on a Picoquant MicroTime 200 instrument operating at an excitation wavelength of 375 nm) of apoA-I fibrils formed with a 2-fold molar excess of heparin doped with 1% w/w of a heparin fluorescein conjugate (ThermoFisher Scientific). Fibrils were washed with aqueous buffer before sedimentation and dispersion onto glass coverslips. The  lower image  shows the total mean fluorescence lifetime corresponding to a best fitting biexponential curve of time constants 1.5 and 4.0 ns.
    Figure Legend Snippet: ApoA-I fibril formation in the absence or presence of heparin. A, negative stain TEM images of apoA-I aggregates formed at pH 4 in the absence of heparin. B , TEM images of apoA-I fibrils formed in the presence of 14–15 kDa of heparin (2-fold m excess). C, distribution and means of fibril widths measured from TEM images of apoA-I ± heparin. D, determination of heparin association with apoA-I fibrils. Left , time course of uronic acid generation resulting from heparin cleavage by heparinase I. Right , calculation of heparin remaining in solution following sedimentation with the protein at different molar ratios. Δ A 232 was measured as the end point A 232 value minus the initial A 232 . All initial solutions contained 1 mg/ml heparin (∼72 μ m ) before the addition of apoA-I and sedimentation of the aggregates formed. The concentrations shown represent the amount of heparin remaining in solution after removal of the insoluble material. E, fluorescence lifetime image (obtained on a Picoquant MicroTime 200 instrument operating at an excitation wavelength of 375 nm) of apoA-I fibrils formed with a 2-fold molar excess of heparin doped with 1% w/w of a heparin fluorescein conjugate (ThermoFisher Scientific). Fibrils were washed with aqueous buffer before sedimentation and dispersion onto glass coverslips. The lower image shows the total mean fluorescence lifetime corresponding to a best fitting biexponential curve of time constants 1.5 and 4.0 ns.

    Techniques Used: Staining, Transmission Electron Microscopy, Sedimentation, Fluorescence

    4) Product Images from "Heparin Promotes Cardiac Differentiation of Human Pluripotent Stem Cells in Chemically Defined Albumin‐Free Medium, Enabling Consistent Manufacture of Cardiomyocytes"

    Article Title: Heparin Promotes Cardiac Differentiation of Human Pluripotent Stem Cells in Chemically Defined Albumin‐Free Medium, Enabling Consistent Manufacture of Cardiomyocytes

    Journal: Stem Cells Translational Medicine

    doi: 10.5966/sctm.2015-0428

    Heparin alone promoted cardiac differentiation from human pluripotent stem cells without treatment of Wnt inhibitors. (A): Immunofluorescence for NKX2.5 (red) and CTNT‐positive cells (green) at day 10 after treatment with 1 μg/ml or 3 μg/ml heparin in the absence or presence of IWP2. Scale bar = 50 μm. (B): Flow cytometry for CTNT (left) and real‐time polymerase chain reaction of NKX2.5 (right) comparing the dosage (treatment from day 1 to day 7) and time course (3 μg/ml treatment) of heparin treatment to promote cardiac differentiation in the absence of IWP2 treatment. Data are presented as the mean ± SD of three or more independent experiments. (C): Immunofluorescence (left) of CTNT (green) and NKX2.5 (red) as well as flow cytometry (right) with CTNT antibody, demonstrating that heparinase I blocked the effects of heparin and inhibited cardiomyocyte derivation. Data are presented as the mean ± SD of three or more independent experiments. Scale bar = 50 μm. (D): Heat map illustrating the promotion of cardiogenesis by heparin during differentiation at day 6 and day 10 in either absence or presence of IWP2. Abbreviations: CTNT, cardiac troponin T; ctrl, control; DAPI, 4′,6‐diamidino‐2‐phenylindole; hep, heparin; hpn, heparinase I.
    Figure Legend Snippet: Heparin alone promoted cardiac differentiation from human pluripotent stem cells without treatment of Wnt inhibitors. (A): Immunofluorescence for NKX2.5 (red) and CTNT‐positive cells (green) at day 10 after treatment with 1 μg/ml or 3 μg/ml heparin in the absence or presence of IWP2. Scale bar = 50 μm. (B): Flow cytometry for CTNT (left) and real‐time polymerase chain reaction of NKX2.5 (right) comparing the dosage (treatment from day 1 to day 7) and time course (3 μg/ml treatment) of heparin treatment to promote cardiac differentiation in the absence of IWP2 treatment. Data are presented as the mean ± SD of three or more independent experiments. (C): Immunofluorescence (left) of CTNT (green) and NKX2.5 (red) as well as flow cytometry (right) with CTNT antibody, demonstrating that heparinase I blocked the effects of heparin and inhibited cardiomyocyte derivation. Data are presented as the mean ± SD of three or more independent experiments. Scale bar = 50 μm. (D): Heat map illustrating the promotion of cardiogenesis by heparin during differentiation at day 6 and day 10 in either absence or presence of IWP2. Abbreviations: CTNT, cardiac troponin T; ctrl, control; DAPI, 4′,6‐diamidino‐2‐phenylindole; hep, heparin; hpn, heparinase I.

    Techniques Used: Immunofluorescence, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction

    Related Articles

    In Vitro:

    Article Title: Epigallocatechin-3-gallate remodels apolipoprotein A-I amyloid fibrils into soluble oligomers in the presence of heparin
    Article Snippet: .. Binding of heparin to apoA-I fibrils In vitro binding assays of apoA-I fibril-heparin binding were performed using an adaptation of a procedure described previously , in which the amount of GAG remaining unbound at different fibril/GAG concentration ratios was determined using Bacteroides heparinase I (New England Biolabs Ltd., UK). .. The heparinase enzyme cleaves heparin yielding oligosaccharide products containing unsaturated uronic acids, which can be detected using UV spectroscopy at 232 nm.

    Concentration Assay:

    Article Title: Epigallocatechin-3-gallate remodels apolipoprotein A-I amyloid fibrils into soluble oligomers in the presence of heparin
    Article Snippet: .. Binding of heparin to apoA-I fibrils In vitro binding assays of apoA-I fibril-heparin binding were performed using an adaptation of a procedure described previously , in which the amount of GAG remaining unbound at different fibril/GAG concentration ratios was determined using Bacteroides heparinase I (New England Biolabs Ltd., UK). .. The heparinase enzyme cleaves heparin yielding oligosaccharide products containing unsaturated uronic acids, which can be detected using UV spectroscopy at 232 nm.

    Incubation:

    Article Title: Heparin affinity purification of extracellular vesicles
    Article Snippet: .. Beads were then washed three times with PBS 1x and either treated with Bacteroides Heparinase I (New England Biolabs, Ipswich, MA; 60U/ml heparin beads) according to the manufacturer’s recommendations or mock treated (incubation buffer devoid of heparinase) for 1 hr at 30 °C. ..

    other:

    Article Title: Heparin Promotes Cardiac Differentiation of Human Pluripotent Stem Cells in Chemically Defined Albumin‐Free Medium, Enabling Consistent Manufacture of Cardiomyocytes
    Article Snippet: The growth factors, inhibitors, and enzymes used in culture were as follows: activin A (10 ng/ml, catalog no. 338‐AC/CF; R & D Systems, Minneapolis, MN, https://www.rndsystems.com ), BMP4 (10 ng/ml, catalog no. 314‐BP/CF; R & D Systems), fibroblast growth factor 2 (FGF2; 100 ng/ml, catalog no. 100‐18B; Peprotech, Rocky Hill, NJ, https://www.peprotech.com ), transforming growth factor β (TGFβ) 1 (1.74 ng/ml, catalog no. 240‐B/CF; R & D), dorsomorphin (3 μM, catalog no. 04‐0024; Stemgent, Lexington, MA, https://www.stemgent.com ), LDN‐193189 (0.1 μM, catalog no. 04‐0074; Stemgent), PD0325901 (1 μM, catalog no. 04‐0008; Stemgent), SB431542 (3 μM, catalog no. s1067; Selleckchem, Houston, TX, http://www.selleckchem.com ), heparin (catalog no. H3149; Sigma‐Aldrich), heparinase I (catalog no. P0735S; New England Biolabs, Ipswich, MA, https://www.neb.com ).

    Liquid Chromatography with Mass Spectroscopy:

    Article Title: Serial in-solution digestion protocol for mass spectrometry-based glycomics and proteomics analysis.
    Article Snippet: .. Advancement in mass spectrometry has revolutionized the field of proteomics. .. Advancement in mass spectrometry has revolutionized the field of proteomics.

    Binding Assay:

    Article Title: Epigallocatechin-3-gallate remodels apolipoprotein A-I amyloid fibrils into soluble oligomers in the presence of heparin
    Article Snippet: .. Binding of heparin to apoA-I fibrils In vitro binding assays of apoA-I fibril-heparin binding were performed using an adaptation of a procedure described previously , in which the amount of GAG remaining unbound at different fibril/GAG concentration ratios was determined using Bacteroides heparinase I (New England Biolabs Ltd., UK). .. The heparinase enzyme cleaves heparin yielding oligosaccharide products containing unsaturated uronic acids, which can be detected using UV spectroscopy at 232 nm.

    Molecular Weight:

    Article Title: Serial in-solution digestion protocol for mass spectrometry-based glycomics and proteomics analysis.
    Article Snippet: .. Advancement in mass spectrometry has revolutionized the field of proteomics. .. Advancement in mass spectrometry has revolutionized the field of proteomics.

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  • 95
    New England Biolabs bacteroides heparinase iii
    Flow cytometry studies indicate a higher uptake of CPP-R57 over CPP-S57 under a variety of conditions. Various cell types were incubated with fluorescently labeled CPP peptides and the uptake measured by flow cytometry. ( a ) 293T cells exposed to 2 μM of a control peptide, CPP-R57 or CPP-S57 peptides followed by analysis by flow cytometry. Proportional MFI values between experiments (n = 4) were then averaged and plotted on a histogram. Raw MFI values for each sample across different experiments spanned the following ranges: control (1.8–5.5), CPP-R57 (41.9–301.0) and CPP-S57 (11.6–99.0). ( b ) 293T cells and THP-1 cells exposed to increasing concentrations of CPPs as indicated to study dose response. ( c ) 293T cells exposed to 2 μM of CPPs at either 37 °C or 4 °C. Data was combined between different experiments (n = 4), and raw MFI values spanned the following ranges: control (2.8–5.3), CPP-R57 (58.8–266.0) and CPP-S57 (20.0–97.3). ( d ) 293T cells were exposed to CPPs at the indicated concentrations of heparin. ( e ) 293T cells treated for 2 h with the indicated concentrations of <t>Heparinase</t> I enzyme (with Heparinase <t>III</t> enzyme included at 1/17 the concentration of Heparinase I), then exposed to 2 μM of CPP. Data was combined between different experiments (n = 2), and raw MFI values spanned the following ranges: control 0 U (2.1–2.3), CPP-R57 0 U (114.0–145.0), CPP-S57 0 U (18.3–31.9), CPP-R57 1.9 U (64.6–75.1), CPP-S57 1.9 U (7.4–10.9), CPP-R57 3.8 U (46.3–49.9), CPP-S57 3.8 U (6.1–6.8), CPP-R57 7.5 U (34.1–35.4), CPP-S57 7.5 U (5.0–5.2), CPP-R57 15 U (27.8–35.2), and CPP-S57 15 U (4.0–5.0) (***p
    Bacteroides Heparinase Iii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bacteroides heparinase iii/product/New England Biolabs
    Average 95 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    bacteroides heparinase iii - by Bioz Stars, 2020-08
    95/100 stars
      Buy from Supplier

    Image Search Results


    Flow cytometry studies indicate a higher uptake of CPP-R57 over CPP-S57 under a variety of conditions. Various cell types were incubated with fluorescently labeled CPP peptides and the uptake measured by flow cytometry. ( a ) 293T cells exposed to 2 μM of a control peptide, CPP-R57 or CPP-S57 peptides followed by analysis by flow cytometry. Proportional MFI values between experiments (n = 4) were then averaged and plotted on a histogram. Raw MFI values for each sample across different experiments spanned the following ranges: control (1.8–5.5), CPP-R57 (41.9–301.0) and CPP-S57 (11.6–99.0). ( b ) 293T cells and THP-1 cells exposed to increasing concentrations of CPPs as indicated to study dose response. ( c ) 293T cells exposed to 2 μM of CPPs at either 37 °C or 4 °C. Data was combined between different experiments (n = 4), and raw MFI values spanned the following ranges: control (2.8–5.3), CPP-R57 (58.8–266.0) and CPP-S57 (20.0–97.3). ( d ) 293T cells were exposed to CPPs at the indicated concentrations of heparin. ( e ) 293T cells treated for 2 h with the indicated concentrations of Heparinase I enzyme (with Heparinase III enzyme included at 1/17 the concentration of Heparinase I), then exposed to 2 μM of CPP. Data was combined between different experiments (n = 2), and raw MFI values spanned the following ranges: control 0 U (2.1–2.3), CPP-R57 0 U (114.0–145.0), CPP-S57 0 U (18.3–31.9), CPP-R57 1.9 U (64.6–75.1), CPP-S57 1.9 U (7.4–10.9), CPP-R57 3.8 U (46.3–49.9), CPP-S57 3.8 U (6.1–6.8), CPP-R57 7.5 U (34.1–35.4), CPP-S57 7.5 U (5.0–5.2), CPP-R57 15 U (27.8–35.2), and CPP-S57 15 U (4.0–5.0) (***p

    Journal: Scientific Reports

    Article Title: A Naturally Occurring Polymorphism in the HIV-1 Tat Basic Domain Inhibits Uptake by Bystander Cells and Leads to Reduced Neuroinflammation

    doi: 10.1038/s41598-019-39531-5

    Figure Lengend Snippet: Flow cytometry studies indicate a higher uptake of CPP-R57 over CPP-S57 under a variety of conditions. Various cell types were incubated with fluorescently labeled CPP peptides and the uptake measured by flow cytometry. ( a ) 293T cells exposed to 2 μM of a control peptide, CPP-R57 or CPP-S57 peptides followed by analysis by flow cytometry. Proportional MFI values between experiments (n = 4) were then averaged and plotted on a histogram. Raw MFI values for each sample across different experiments spanned the following ranges: control (1.8–5.5), CPP-R57 (41.9–301.0) and CPP-S57 (11.6–99.0). ( b ) 293T cells and THP-1 cells exposed to increasing concentrations of CPPs as indicated to study dose response. ( c ) 293T cells exposed to 2 μM of CPPs at either 37 °C or 4 °C. Data was combined between different experiments (n = 4), and raw MFI values spanned the following ranges: control (2.8–5.3), CPP-R57 (58.8–266.0) and CPP-S57 (20.0–97.3). ( d ) 293T cells were exposed to CPPs at the indicated concentrations of heparin. ( e ) 293T cells treated for 2 h with the indicated concentrations of Heparinase I enzyme (with Heparinase III enzyme included at 1/17 the concentration of Heparinase I), then exposed to 2 μM of CPP. Data was combined between different experiments (n = 2), and raw MFI values spanned the following ranges: control 0 U (2.1–2.3), CPP-R57 0 U (114.0–145.0), CPP-S57 0 U (18.3–31.9), CPP-R57 1.9 U (64.6–75.1), CPP-S57 1.9 U (7.4–10.9), CPP-R57 3.8 U (46.3–49.9), CPP-S57 3.8 U (6.1–6.8), CPP-R57 7.5 U (34.1–35.4), CPP-S57 7.5 U (5.0–5.2), CPP-R57 15 U (27.8–35.2), and CPP-S57 15 U (4.0–5.0) (***p

    Article Snippet: The cell surface HSPGs were digested using Heparinase I and Heparinase III enzymes (both cloned from Bacteroides strains; NE Biolabs #P0735S and #P0737S respectively).

    Techniques: Flow Cytometry, Cytometry, Conditioned Place Preference, Incubation, Labeling, Concentration Assay

    Extracellular vesicles are efficiently isolated and purified using heparin-coated agarose beads. (a) Heparin coated agarose beads are incubated with EVs released from a variety of cells lines, (i), to yield an EV/heparin complex, (ii). Free floating proteins and nucleic acids are washed away with PBS, (iii). Beads are the incubated overnight with 2.15 M NaCl and the EVs are released and collected by spinning down the beads and collecting the supernatant (iv). Collected EVs are used as a source of RNA (biomarker) or used in biological assays (v ). (b) Nanoparticle tracking analysis (NTA) counts of heparin-purified human 293T-derived EVs eluted with 2.15 M NaCl overnight at 4 °C following 3 wash steps. ( c ) To show specific heparin affinity we incubated heparin beads overnight with EVs, then rinsed beads 3 times with PBS and treated with Bacteroides Heparinase I or incubation buffer without heparinase and fractions were analyzed by NTA. ( d ) EVs were mixed with heparin beads and one round of purification was performed. The unbound and eluted fractions from round one were separately incubated with a fresh batch of heparin beads and round 2 purification performed on these samples. NTA was performed on each fraction of round 2 purification.

    Journal: Scientific Reports

    Article Title: Heparin affinity purification of extracellular vesicles

    doi: 10.1038/srep10266

    Figure Lengend Snippet: Extracellular vesicles are efficiently isolated and purified using heparin-coated agarose beads. (a) Heparin coated agarose beads are incubated with EVs released from a variety of cells lines, (i), to yield an EV/heparin complex, (ii). Free floating proteins and nucleic acids are washed away with PBS, (iii). Beads are the incubated overnight with 2.15 M NaCl and the EVs are released and collected by spinning down the beads and collecting the supernatant (iv). Collected EVs are used as a source of RNA (biomarker) or used in biological assays (v ). (b) Nanoparticle tracking analysis (NTA) counts of heparin-purified human 293T-derived EVs eluted with 2.15 M NaCl overnight at 4 °C following 3 wash steps. ( c ) To show specific heparin affinity we incubated heparin beads overnight with EVs, then rinsed beads 3 times with PBS and treated with Bacteroides Heparinase I or incubation buffer without heparinase and fractions were analyzed by NTA. ( d ) EVs were mixed with heparin beads and one round of purification was performed. The unbound and eluted fractions from round one were separately incubated with a fresh batch of heparin beads and round 2 purification performed on these samples. NTA was performed on each fraction of round 2 purification.

    Article Snippet: Beads were then washed three times with PBS 1x and either treated with Bacteroides Heparinase I (New England Biolabs, Ipswich, MA; 60U/ml heparin beads) according to the manufacturer’s recommendations or mock treated (incubation buffer devoid of heparinase) for 1 hr at 30 °C.

    Techniques: Isolation, Purification, Incubation, Biomarker Assay, Derivative Assay

    ApoA-I fibril formation in the absence or presence of heparin. A,  negative stain TEM images of apoA-I aggregates formed at pH 4 in the absence of heparin.  B , TEM images of apoA-I fibrils formed in the presence of 14–15 kDa of heparin (2-fold  m  excess).  C,  distribution and means of fibril widths measured from TEM images of apoA-I ± heparin.  D,  determination of heparin association with apoA-I fibrils.  Left , time course of uronic acid generation resulting from heparin cleavage by heparinase I.  Right , calculation of heparin remaining in solution following sedimentation with the protein at different molar ratios. Δ A 232  was measured as the end point  A 232  value minus the initial  A 232 . All initial solutions contained 1 mg/ml heparin (∼72 μ m ) before the addition of apoA-I and sedimentation of the aggregates formed. The concentrations shown represent the amount of heparin remaining in solution after removal of the insoluble material.  E,  fluorescence lifetime image (obtained on a Picoquant MicroTime 200 instrument operating at an excitation wavelength of 375 nm) of apoA-I fibrils formed with a 2-fold molar excess of heparin doped with 1% w/w of a heparin fluorescein conjugate (ThermoFisher Scientific). Fibrils were washed with aqueous buffer before sedimentation and dispersion onto glass coverslips. The  lower image  shows the total mean fluorescence lifetime corresponding to a best fitting biexponential curve of time constants 1.5 and 4.0 ns.

    Journal: The Journal of Biological Chemistry

    Article Title: Epigallocatechin-3-gallate remodels apolipoprotein A-I amyloid fibrils into soluble oligomers in the presence of heparin

    doi: 10.1074/jbc.RA118.002038

    Figure Lengend Snippet: ApoA-I fibril formation in the absence or presence of heparin. A, negative stain TEM images of apoA-I aggregates formed at pH 4 in the absence of heparin. B , TEM images of apoA-I fibrils formed in the presence of 14–15 kDa of heparin (2-fold m excess). C, distribution and means of fibril widths measured from TEM images of apoA-I ± heparin. D, determination of heparin association with apoA-I fibrils. Left , time course of uronic acid generation resulting from heparin cleavage by heparinase I. Right , calculation of heparin remaining in solution following sedimentation with the protein at different molar ratios. Δ A 232 was measured as the end point A 232 value minus the initial A 232 . All initial solutions contained 1 mg/ml heparin (∼72 μ m ) before the addition of apoA-I and sedimentation of the aggregates formed. The concentrations shown represent the amount of heparin remaining in solution after removal of the insoluble material. E, fluorescence lifetime image (obtained on a Picoquant MicroTime 200 instrument operating at an excitation wavelength of 375 nm) of apoA-I fibrils formed with a 2-fold molar excess of heparin doped with 1% w/w of a heparin fluorescein conjugate (ThermoFisher Scientific). Fibrils were washed with aqueous buffer before sedimentation and dispersion onto glass coverslips. The lower image shows the total mean fluorescence lifetime corresponding to a best fitting biexponential curve of time constants 1.5 and 4.0 ns.

    Article Snippet: Binding of heparin to apoA-I fibrils In vitro binding assays of apoA-I fibril-heparin binding were performed using an adaptation of a procedure described previously , in which the amount of GAG remaining unbound at different fibril/GAG concentration ratios was determined using Bacteroides heparinase I (New England Biolabs Ltd., UK).

    Techniques: Staining, Transmission Electron Microscopy, Sedimentation, Fluorescence

    Heparin alone promoted cardiac differentiation from human pluripotent stem cells without treatment of Wnt inhibitors. (A): Immunofluorescence for NKX2.5 (red) and CTNT‐positive cells (green) at day 10 after treatment with 1 μg/ml or 3 μg/ml heparin in the absence or presence of IWP2. Scale bar = 50 μm. (B): Flow cytometry for CTNT (left) and real‐time polymerase chain reaction of NKX2.5 (right) comparing the dosage (treatment from day 1 to day 7) and time course (3 μg/ml treatment) of heparin treatment to promote cardiac differentiation in the absence of IWP2 treatment. Data are presented as the mean ± SD of three or more independent experiments. (C): Immunofluorescence (left) of CTNT (green) and NKX2.5 (red) as well as flow cytometry (right) with CTNT antibody, demonstrating that heparinase I blocked the effects of heparin and inhibited cardiomyocyte derivation. Data are presented as the mean ± SD of three or more independent experiments. Scale bar = 50 μm. (D): Heat map illustrating the promotion of cardiogenesis by heparin during differentiation at day 6 and day 10 in either absence or presence of IWP2. Abbreviations: CTNT, cardiac troponin T; ctrl, control; DAPI, 4′,6‐diamidino‐2‐phenylindole; hep, heparin; hpn, heparinase I.

    Journal: Stem Cells Translational Medicine

    Article Title: Heparin Promotes Cardiac Differentiation of Human Pluripotent Stem Cells in Chemically Defined Albumin‐Free Medium, Enabling Consistent Manufacture of Cardiomyocytes

    doi: 10.5966/sctm.2015-0428

    Figure Lengend Snippet: Heparin alone promoted cardiac differentiation from human pluripotent stem cells without treatment of Wnt inhibitors. (A): Immunofluorescence for NKX2.5 (red) and CTNT‐positive cells (green) at day 10 after treatment with 1 μg/ml or 3 μg/ml heparin in the absence or presence of IWP2. Scale bar = 50 μm. (B): Flow cytometry for CTNT (left) and real‐time polymerase chain reaction of NKX2.5 (right) comparing the dosage (treatment from day 1 to day 7) and time course (3 μg/ml treatment) of heparin treatment to promote cardiac differentiation in the absence of IWP2 treatment. Data are presented as the mean ± SD of three or more independent experiments. (C): Immunofluorescence (left) of CTNT (green) and NKX2.5 (red) as well as flow cytometry (right) with CTNT antibody, demonstrating that heparinase I blocked the effects of heparin and inhibited cardiomyocyte derivation. Data are presented as the mean ± SD of three or more independent experiments. Scale bar = 50 μm. (D): Heat map illustrating the promotion of cardiogenesis by heparin during differentiation at day 6 and day 10 in either absence or presence of IWP2. Abbreviations: CTNT, cardiac troponin T; ctrl, control; DAPI, 4′,6‐diamidino‐2‐phenylindole; hep, heparin; hpn, heparinase I.

    Article Snippet: The growth factors, inhibitors, and enzymes used in culture were as follows: activin A (10 ng/ml, catalog no. 338‐AC/CF; R & D Systems, Minneapolis, MN, https://www.rndsystems.com ), BMP4 (10 ng/ml, catalog no. 314‐BP/CF; R & D Systems), fibroblast growth factor 2 (FGF2; 100 ng/ml, catalog no. 100‐18B; Peprotech, Rocky Hill, NJ, https://www.peprotech.com ), transforming growth factor β (TGFβ) 1 (1.74 ng/ml, catalog no. 240‐B/CF; R & D), dorsomorphin (3 μM, catalog no. 04‐0024; Stemgent, Lexington, MA, https://www.stemgent.com ), LDN‐193189 (0.1 μM, catalog no. 04‐0074; Stemgent), PD0325901 (1 μM, catalog no. 04‐0008; Stemgent), SB431542 (3 μM, catalog no. s1067; Selleckchem, Houston, TX, http://www.selleckchem.com ), heparin (catalog no. H3149; Sigma‐Aldrich), heparinase I (catalog no. P0735S; New England Biolabs, Ipswich, MA, https://www.neb.com ).

    Techniques: Immunofluorescence, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction