α2 3 neuraminidase  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    Name:
    alpha 2 3 Neuraminidase S
    Description:
    alpha 2 3 Neuraminidase S 2 000 units
    Catalog Number:
    P0743L
    Price:
    276
    Category:
    Glycosidases
    Size:
    2 000 units
    Buy from Supplier


    Structured Review

    New England Biolabs α2 3 neuraminidase
    alpha 2 3 Neuraminidase S
    alpha 2 3 Neuraminidase S 2 000 units
    https://www.bioz.com/result/α2 3 neuraminidase/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    α2 3 neuraminidase - by Bioz Stars, 2021-09
    95/100 stars

    Images

    1) Product Images from "Isomeric Separation of N-Glycopeptides Derived from Glycoproteins by Porous Graphitic Carbon (PGC) LC-MS/MS"

    Article Title: Isomeric Separation of N-Glycopeptides Derived from Glycoproteins by Porous Graphitic Carbon (PGC) LC-MS/MS

    Journal: Analytical chemistry

    doi: 10.1021/acs.analchem.0c00668

    EICs of A2G2S2, A2G2S1 and A2G2 glycopeptides in non-treated (a-c) , α2–3 neuraminidase treated (d-f) and α2–3,6,8,9 neuraminidase treated samples (g-i) . The peaks at 37.3 min and 39.2 min in (a) showed sensitivity for α2–3 neuraminidase, which disappeared after enzymatic digestion (d) , indicating these two peaks were containing at least one sialic acid that were α2–3 linked to galactose. Upon α2–3 neuraminidase digestion, A2G2S1 can be detected (e) , which were not originally in sample (b) . Since the 35.5 min peak in (a) remained after α2–3 neuraminidase digestion (d) , both the sialic acid linkages can be assigned α2–6. Comparing the intensities between (e) and (f) , the 37.3 min peak in (a) .
    Figure Legend Snippet: EICs of A2G2S2, A2G2S1 and A2G2 glycopeptides in non-treated (a-c) , α2–3 neuraminidase treated (d-f) and α2–3,6,8,9 neuraminidase treated samples (g-i) . The peaks at 37.3 min and 39.2 min in (a) showed sensitivity for α2–3 neuraminidase, which disappeared after enzymatic digestion (d) , indicating these two peaks were containing at least one sialic acid that were α2–3 linked to galactose. Upon α2–3 neuraminidase digestion, A2G2S1 can be detected (e) , which were not originally in sample (b) . Since the 35.5 min peak in (a) remained after α2–3 neuraminidase digestion (d) , both the sialic acid linkages can be assigned α2–6. Comparing the intensities between (e) and (f) , the 37.3 min peak in (a) .

    Techniques Used:

    2) Product Images from "Alterations of the Human Skin N- and O-Glycome in Basal Cell Carcinoma and Squamous Cell Carcinoma"

    Article Title: Alterations of the Human Skin N- and O-Glycome in Basal Cell Carcinoma and Squamous Cell Carcinoma

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2018.00070

    N -glycosylation changes in squamous cell carcinoma (SCC). (A) Supervised hierarchical cluster analysis of healthy and tumor skin. Rows display each of the 15 patient glycan data (T, tumor tissue; C, healthy control). Columns indicate the N -glycan ID. Five clusters can be observed: in healthy tissue N -glycans with α2-3 linked N -acetylneuraminic acid (NeuAc) appeared to be present in increased levels whereas α2-6-NeuAc and oligomannose N -glycan levels were higher in tumor tissue. (B) Statistical evaluation of sialylated and oligomannose N -glycans uncovered significant changes [ p ≤ 0.04, using a t -test, indicated by an asterisk (*)]. Oligomannose N -glycans were upregulated whereas α2-3 linked NeuAc carrying N -glycans were down regulated in SCC.
    Figure Legend Snippet: N -glycosylation changes in squamous cell carcinoma (SCC). (A) Supervised hierarchical cluster analysis of healthy and tumor skin. Rows display each of the 15 patient glycan data (T, tumor tissue; C, healthy control). Columns indicate the N -glycan ID. Five clusters can be observed: in healthy tissue N -glycans with α2-3 linked N -acetylneuraminic acid (NeuAc) appeared to be present in increased levels whereas α2-6-NeuAc and oligomannose N -glycan levels were higher in tumor tissue. (B) Statistical evaluation of sialylated and oligomannose N -glycans uncovered significant changes [ p ≤ 0.04, using a t -test, indicated by an asterisk (*)]. Oligomannose N -glycans were upregulated whereas α2-3 linked NeuAc carrying N -glycans were down regulated in SCC.

    Techniques Used:

    Healthy human skin N -glycome. (A) Bean diagram representing the 10 most abundant N -glycans determined from 14 patients. Green bars indicate individual data points, the black line represents the median, and the gray area depicts the data density. Columns indicate the glycan structures given by their glycan ID (Table S1 in Supplementary Material). (B) Relative N -glycan class abundances found in healthy skin biopsies. Sialylated and fucosylated structures were the major components representing the human skin N -glycome. (C) Relative abundances of different structure features found on sialylated N -glycans. Blue bars represent sialylated N -glycans with and without core fucose depending on their sialic acid linkage, showing that core fucosylation was a more abundant feature on N -glycans carrying one or two α2-3 linked N -acetylneuraminic acid (NeuAc) residues (if both, α2-3 and α2-6 linkages were present on one N- glycan, this N -glycan was considered in both linkage categories). Overall, α2-6-linked NeuAc was a slightly more abundant feature compared with α2-3-linked NeuAc. Most of the sialylated N -glycans carried two NeuAc residues. Tri-antennary species were below 3% and rather low abundant using porous graphitized carbon, while multiplexed capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF) detected slightly higher levels of tri- and tetra-antennary N -glycans (see Supplementary file “xCGE-LIF quant.xlsx” in Supplementary Material).
    Figure Legend Snippet: Healthy human skin N -glycome. (A) Bean diagram representing the 10 most abundant N -glycans determined from 14 patients. Green bars indicate individual data points, the black line represents the median, and the gray area depicts the data density. Columns indicate the glycan structures given by their glycan ID (Table S1 in Supplementary Material). (B) Relative N -glycan class abundances found in healthy skin biopsies. Sialylated and fucosylated structures were the major components representing the human skin N -glycome. (C) Relative abundances of different structure features found on sialylated N -glycans. Blue bars represent sialylated N -glycans with and without core fucose depending on their sialic acid linkage, showing that core fucosylation was a more abundant feature on N -glycans carrying one or two α2-3 linked N -acetylneuraminic acid (NeuAc) residues (if both, α2-3 and α2-6 linkages were present on one N- glycan, this N -glycan was considered in both linkage categories). Overall, α2-6-linked NeuAc was a slightly more abundant feature compared with α2-3-linked NeuAc. Most of the sialylated N -glycans carried two NeuAc residues. Tri-antennary species were below 3% and rather low abundant using porous graphitized carbon, while multiplexed capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF) detected slightly higher levels of tri- and tetra-antennary N -glycans (see Supplementary file “xCGE-LIF quant.xlsx” in Supplementary Material).

    Techniques Used: Nucleic Acid Electrophoresis, Fluorescence

    3) Product Images from "Glycosylation of Human IgA Directly Inhibits Influenza A and Other Sialic-Acid-Binding Viruses"

    Article Title: Glycosylation of Human IgA Directly Inhibits Influenza A and Other Sialic-Acid-Binding Viruses

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2018.03.027

    Impact of Fc Glycosylation (A) Impact of Fc glycosylation on neutralizing activity. Comparison of the neutralizing activities of wild-type IgG1, wild-type IgA1, and IgA1 in which the glycosylation consensus sequences at position 263 or 459 have been removed. (B) Comparison of the complex glycoforms of IgG1 and IgA1 and the oligomannose-type glycoform of IgA1 expressed in 293S cells (IgA S). (C) Impact of neuraminidase treatment on the neutralization of rg-A/Chicken/Vietnam/C58/2004 (H5N3). The neutralizing activity of the indicated antibodies incubated either with α2–3 neuraminidase from Salmonella typhimurium LT2 (NA S) or α2–3,6,8,9 neuraminidase A from Arthrobacter ureafaciens (NA A) was compared to that of the mock-treated IgA1 and IgG1 molecules. (D) Impact of Fc glycosylation on hemagglutination inhibition. A constant amount of the indicated virus was incubated with titrated amounts of the indicated antibodies and added to chicken erythrocytes that were then allowed to sediment at room temperature. (E) Impact of prolonged incubation with virus. Virus and titrated amounts of the indicated isotype of mAb 3.1 were incubated for either 1 hr or overnight before residual infectivity was determined.
    Figure Legend Snippet: Impact of Fc Glycosylation (A) Impact of Fc glycosylation on neutralizing activity. Comparison of the neutralizing activities of wild-type IgG1, wild-type IgA1, and IgA1 in which the glycosylation consensus sequences at position 263 or 459 have been removed. (B) Comparison of the complex glycoforms of IgG1 and IgA1 and the oligomannose-type glycoform of IgA1 expressed in 293S cells (IgA S). (C) Impact of neuraminidase treatment on the neutralization of rg-A/Chicken/Vietnam/C58/2004 (H5N3). The neutralizing activity of the indicated antibodies incubated either with α2–3 neuraminidase from Salmonella typhimurium LT2 (NA S) or α2–3,6,8,9 neuraminidase A from Arthrobacter ureafaciens (NA A) was compared to that of the mock-treated IgA1 and IgG1 molecules. (D) Impact of Fc glycosylation on hemagglutination inhibition. A constant amount of the indicated virus was incubated with titrated amounts of the indicated antibodies and added to chicken erythrocytes that were then allowed to sediment at room temperature. (E) Impact of prolonged incubation with virus. Virus and titrated amounts of the indicated isotype of mAb 3.1 were incubated for either 1 hr or overnight before residual infectivity was determined.

    Techniques Used: Activity Assay, Neutralization, Incubation, HI Assay, Infection

    4) Product Images from "Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1"

    Article Title: Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32534-8

    SPR analysis of bindings of lectin and SKM9-2 to glycosidase-treated SKMepmin3. Purified SKMepmin3 was incubated with α2-3 Neuraminidase S, Neuraminidase A, or Neuraminidase A + O -Glycosidase; and then was immobilized as a ligand on Ni 2+ -binding sensor chip NTA. The analytes were used at a concentration of 5 µg/mL. ( a ) ABA, ( b ) Jacalin, ( c ) rACG, ( d ) SKM9-2. Glycosidase-digested sample was not tested in the experiment with rACG.
    Figure Legend Snippet: SPR analysis of bindings of lectin and SKM9-2 to glycosidase-treated SKMepmin3. Purified SKMepmin3 was incubated with α2-3 Neuraminidase S, Neuraminidase A, or Neuraminidase A + O -Glycosidase; and then was immobilized as a ligand on Ni 2+ -binding sensor chip NTA. The analytes were used at a concentration of 5 µg/mL. ( a ) ABA, ( b ) Jacalin, ( c ) rACG, ( d ) SKM9-2. Glycosidase-digested sample was not tested in the experiment with rACG.

    Techniques Used: SPR Assay, Purification, Incubation, Binding Assay, Chromatin Immunoprecipitation, Concentration Assay

    SKM9-2 epitope produced from mesothelioma cells. ( a ) Chromatogram of SKMepmin3 separation using anion exchange column. SKMepmin3 produced from HEK293T (blue line) or ACC-MESO4 (pink line) was affinity-purified using SKM9-2-immobilized resins and separated by Mini Q 4.6/50 PE. A major peak of SKMepmin3 purified from HEK293T contained two disialyl T antigens (Fig. 6 ). ( b ) Western blotting for epitope-fused GPI-anchor protein (7.6231) treated with neuraminidases. Cell lysate of 7.6231-transfected ACC-MESO1 (top panel), ACC-MESO4 (middle panel), or HEK293T (lower panel) was treated with α2-3 Neuraminidase S or Neuraminidase A, resolved by 4–15% SDS-PAGE, and analyzed by western blotting using SKM9-2. ( c ) Western blotting for full-length HEG1 treated with neuraminidases. Cell lysates of ACC-MESO4 (upper panel) and HEG1-transfected HEK293T (lower panel) were treated with α2-3 Neuraminidase S or Neuraminidase A and analyzed by 6% SDS-PAGE and western blotting using SKM9-2. ( d ) Schematic of SKM9-2 epitope. Full-length blots are presented in Supplementary Fig. S10 .
    Figure Legend Snippet: SKM9-2 epitope produced from mesothelioma cells. ( a ) Chromatogram of SKMepmin3 separation using anion exchange column. SKMepmin3 produced from HEK293T (blue line) or ACC-MESO4 (pink line) was affinity-purified using SKM9-2-immobilized resins and separated by Mini Q 4.6/50 PE. A major peak of SKMepmin3 purified from HEK293T contained two disialyl T antigens (Fig. 6 ). ( b ) Western blotting for epitope-fused GPI-anchor protein (7.6231) treated with neuraminidases. Cell lysate of 7.6231-transfected ACC-MESO1 (top panel), ACC-MESO4 (middle panel), or HEK293T (lower panel) was treated with α2-3 Neuraminidase S or Neuraminidase A, resolved by 4–15% SDS-PAGE, and analyzed by western blotting using SKM9-2. ( c ) Western blotting for full-length HEG1 treated with neuraminidases. Cell lysates of ACC-MESO4 (upper panel) and HEG1-transfected HEK293T (lower panel) were treated with α2-3 Neuraminidase S or Neuraminidase A and analyzed by 6% SDS-PAGE and western blotting using SKM9-2. ( d ) Schematic of SKM9-2 epitope. Full-length blots are presented in Supplementary Fig. S10 .

    Techniques Used: Produced, Affinity Purification, Purification, Western Blot, Transfection, SDS Page

    5) Product Images from "Glycosylation of Human IgA Directly Inhibits Influenza A and Other Sialic-Acid-Binding Viruses"

    Article Title: Glycosylation of Human IgA Directly Inhibits Influenza A and Other Sialic-Acid-Binding Viruses

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2018.03.027

    Impact of Fc Glycosylation (A) Impact of Fc glycosylation on neutralizing activity. Comparison of the neutralizing activities of wild-type IgG1, wild-type IgA1, and IgA1 in which the glycosylation consensus sequences at position 263 or 459 have been removed. (B) Comparison of the complex glycoforms of IgG1 and IgA1 and the oligomannose-type glycoform of IgA1 expressed in 293S cells (IgA S). (C) Impact of neuraminidase treatment on the neutralization of rg-A/Chicken/Vietnam/C58/2004 (H5N3). The neutralizing activity of the indicated antibodies incubated either with α2–3 neuraminidase from Salmonella typhimurium LT2 (NA S) or α2–3,6,8,9 neuraminidase A from Arthrobacter ureafaciens (NA A) was compared to that of the mock-treated IgA1 and IgG1 molecules. (D) Impact of Fc glycosylation on hemagglutination inhibition. A constant amount of the indicated virus was incubated with titrated amounts of the indicated antibodies and added to chicken erythrocytes that were then allowed to sediment at room temperature. (E) Impact of prolonged incubation with virus. Virus and titrated amounts of the indicated isotype of mAb 3.1 were incubated for either 1 hr or overnight before residual infectivity was determined.
    Figure Legend Snippet: Impact of Fc Glycosylation (A) Impact of Fc glycosylation on neutralizing activity. Comparison of the neutralizing activities of wild-type IgG1, wild-type IgA1, and IgA1 in which the glycosylation consensus sequences at position 263 or 459 have been removed. (B) Comparison of the complex glycoforms of IgG1 and IgA1 and the oligomannose-type glycoform of IgA1 expressed in 293S cells (IgA S). (C) Impact of neuraminidase treatment on the neutralization of rg-A/Chicken/Vietnam/C58/2004 (H5N3). The neutralizing activity of the indicated antibodies incubated either with α2–3 neuraminidase from Salmonella typhimurium LT2 (NA S) or α2–3,6,8,9 neuraminidase A from Arthrobacter ureafaciens (NA A) was compared to that of the mock-treated IgA1 and IgG1 molecules. (D) Impact of Fc glycosylation on hemagglutination inhibition. A constant amount of the indicated virus was incubated with titrated amounts of the indicated antibodies and added to chicken erythrocytes that were then allowed to sediment at room temperature. (E) Impact of prolonged incubation with virus. Virus and titrated amounts of the indicated isotype of mAb 3.1 were incubated for either 1 hr or overnight before residual infectivity was determined.

    Techniques Used: Activity Assay, Neutralization, Incubation, HI Assay, Infection

    6) Product Images from "Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1"

    Article Title: Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32534-8

    SPR analysis of bindings of lectin and SKM9-2 to glycosidase-treated SKMepmin3. Purified SKMepmin3 was incubated with α2-3 Neuraminidase S, Neuraminidase A, or Neuraminidase A + O -Glycosidase; and then was immobilized as a ligand on Ni 2+ -binding sensor chip NTA. The analytes were used at a concentration of 5 µg/mL. ( a ) ABA, ( b ) Jacalin, ( c ) rACG, ( d ) SKM9-2. Glycosidase-digested sample was not tested in the experiment with rACG.
    Figure Legend Snippet: SPR analysis of bindings of lectin and SKM9-2 to glycosidase-treated SKMepmin3. Purified SKMepmin3 was incubated with α2-3 Neuraminidase S, Neuraminidase A, or Neuraminidase A + O -Glycosidase; and then was immobilized as a ligand on Ni 2+ -binding sensor chip NTA. The analytes were used at a concentration of 5 µg/mL. ( a ) ABA, ( b ) Jacalin, ( c ) rACG, ( d ) SKM9-2. Glycosidase-digested sample was not tested in the experiment with rACG.

    Techniques Used: SPR Assay, Purification, Incubation, Binding Assay, Chromatin Immunoprecipitation, Concentration Assay

    SKM9-2 epitope produced from mesothelioma cells. ( a ) Chromatogram of SKMepmin3 separation using anion exchange column. SKMepmin3 produced from HEK293T (blue line) or ACC-MESO4 (pink line) was affinity-purified using SKM9-2-immobilized resins and separated by Mini Q 4.6/50 PE. A major peak of SKMepmin3 purified from HEK293T contained two disialyl T antigens (Fig. 6 ). ( b ) Western blotting for epitope-fused GPI-anchor protein (7.6231) treated with neuraminidases. Cell lysate of 7.6231-transfected ACC-MESO1 (top panel), ACC-MESO4 (middle panel), or HEK293T (lower panel) was treated with α2-3 Neuraminidase S or Neuraminidase A, resolved by 4–15% SDS-PAGE, and analyzed by western blotting using SKM9-2. ( c ) Western blotting for full-length HEG1 treated with neuraminidases. Cell lysates of ACC-MESO4 (upper panel) and HEG1-transfected HEK293T (lower panel) were treated with α2-3 Neuraminidase S or Neuraminidase A and analyzed by 6% SDS-PAGE and western blotting using SKM9-2. ( d ) Schematic of SKM9-2 epitope. Full-length blots are presented in Supplementary Fig. S10 .
    Figure Legend Snippet: SKM9-2 epitope produced from mesothelioma cells. ( a ) Chromatogram of SKMepmin3 separation using anion exchange column. SKMepmin3 produced from HEK293T (blue line) or ACC-MESO4 (pink line) was affinity-purified using SKM9-2-immobilized resins and separated by Mini Q 4.6/50 PE. A major peak of SKMepmin3 purified from HEK293T contained two disialyl T antigens (Fig. 6 ). ( b ) Western blotting for epitope-fused GPI-anchor protein (7.6231) treated with neuraminidases. Cell lysate of 7.6231-transfected ACC-MESO1 (top panel), ACC-MESO4 (middle panel), or HEK293T (lower panel) was treated with α2-3 Neuraminidase S or Neuraminidase A, resolved by 4–15% SDS-PAGE, and analyzed by western blotting using SKM9-2. ( c ) Western blotting for full-length HEG1 treated with neuraminidases. Cell lysates of ACC-MESO4 (upper panel) and HEG1-transfected HEK293T (lower panel) were treated with α2-3 Neuraminidase S or Neuraminidase A and analyzed by 6% SDS-PAGE and western blotting using SKM9-2. ( d ) Schematic of SKM9-2 epitope. Full-length blots are presented in Supplementary Fig. S10 .

    Techniques Used: Produced, Affinity Purification, Purification, Western Blot, Transfection, SDS Page

    7) Product Images from "Human mastadenovirus-B (HAdV-B)-specific E3-CR1β and E3-CR1γ glycoproteins interact with each other and localize at the plasma membrane of non-polarized airway epithelial cells"

    Article Title: Human mastadenovirus-B (HAdV-B)-specific E3-CR1β and E3-CR1γ glycoproteins interact with each other and localize at the plasma membrane of non-polarized airway epithelial cells

    Journal: Virology

    doi: 10.1016/j.virol.2020.04.005

    HAdV-3 E3-20.1K is N-glycosylated whereas E3-20.5K is N-and O-glycosylated. A549 cells were infected with the HAdV-3 N-tag wt virus at a MOI of 10 pfu/cell. At 48 hpi cell lysates were prepared and treated for 1 h with PNGase F, or α2-3,6,8,9 neuraminidase A, or O-glycosidase either individually or sequentially with α2-3,6,8,9 neuraminidase A and o-glycosidase or with PNGase F, α2-3,6,8,9 neuraminidase A, and o-glycosidase in the order mentioned. After treatment, the lysates were analyzed for the expression of VSV-G E3-20.1K, HA E3-20.5K, and GAPDH (loading control) by SDS-PAGE and WB. The blot is representative of three independent experiments.
    Figure Legend Snippet: HAdV-3 E3-20.1K is N-glycosylated whereas E3-20.5K is N-and O-glycosylated. A549 cells were infected with the HAdV-3 N-tag wt virus at a MOI of 10 pfu/cell. At 48 hpi cell lysates were prepared and treated for 1 h with PNGase F, or α2-3,6,8,9 neuraminidase A, or O-glycosidase either individually or sequentially with α2-3,6,8,9 neuraminidase A and o-glycosidase or with PNGase F, α2-3,6,8,9 neuraminidase A, and o-glycosidase in the order mentioned. After treatment, the lysates were analyzed for the expression of VSV-G E3-20.1K, HA E3-20.5K, and GAPDH (loading control) by SDS-PAGE and WB. The blot is representative of three independent experiments.

    Techniques Used: Infection, Expressing, SDS Page, Western Blot

    8) Product Images from "An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides"

    Article Title: An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

    Journal: Nature Communications

    doi: 10.1038/ncomms15487

    High-salt conditions increase complex N-glycomolecule binding to wt Fbs1. ( a ) The presence of 2 M NaCl increases SGP-TMR binding to wt Fbs1 in an N-glycan-dependent manner. PNGase F (+) indicates SGP-TMR was pretreated with PNGase F to cleave the glycan from the fluorophore-labelled peptide (sequence KVANKT). SGP-TMR with or without PNGase F treatment was incubated with Fbs1 beads in low-salt (LS) conditions or high-salt (HS) conditions. SGP-TMR binding to Fbs1 beads was measured, and affinity to Fbs1 is indicated by percentage of recovery (amount of bound SGP-TMR/amount of input SGP-TMR). Results represent the mean±s.e.m. of three replicates. ( b ) HS conditions increase Fbs1 binding to sialylated fetuin relative to RNase B, which contains high-mannose N-glycans. A mixture of denatured fetuin and RNase B was subjected to an Fbs1 bead pulldown assay. Lane 1 indicates the input ratio of fetuin to RNase B. Lanes 2 and 3 show the amounts of fetuin and RNase B pulled down by Fbs1 beads in LS and HS conditions. Asterisk denotes a small amount of SNAP-Fbs1 that leaches from the Fbs1 beads. N-glycan structures present within fetuin and RNase B are illustrated. A representative SDS–PAGE gel is shown from two experiments. ( c ) Reciprocal pulldown of SNAP-Fbs1 by denatured fetuin or RNase B beads in LS or HS conditions. A representative SDS–PAGE gel is shown from two experiments. ( d ) HS conditions have no effect on Fbs1 binding to asialo-SGP-TMR. SGP-TMR was trimmed with α2-3,6,8 Neuraminidase to produce asialo-SGP-TMR (structures shown in Fig. 1d , glycopeptide 1 and 2). SGP-TMR and asialo-SGP-TMR were incubated with Fbs1 beads in LS buffer or HS buffer. SGP-TMR or asialo-SGP-TMR relative affinity to Fbs1 is indicated by the recovery percentage. Results represent the mean±s.e.m. of three replicates.
    Figure Legend Snippet: High-salt conditions increase complex N-glycomolecule binding to wt Fbs1. ( a ) The presence of 2 M NaCl increases SGP-TMR binding to wt Fbs1 in an N-glycan-dependent manner. PNGase F (+) indicates SGP-TMR was pretreated with PNGase F to cleave the glycan from the fluorophore-labelled peptide (sequence KVANKT). SGP-TMR with or without PNGase F treatment was incubated with Fbs1 beads in low-salt (LS) conditions or high-salt (HS) conditions. SGP-TMR binding to Fbs1 beads was measured, and affinity to Fbs1 is indicated by percentage of recovery (amount of bound SGP-TMR/amount of input SGP-TMR). Results represent the mean±s.e.m. of three replicates. ( b ) HS conditions increase Fbs1 binding to sialylated fetuin relative to RNase B, which contains high-mannose N-glycans. A mixture of denatured fetuin and RNase B was subjected to an Fbs1 bead pulldown assay. Lane 1 indicates the input ratio of fetuin to RNase B. Lanes 2 and 3 show the amounts of fetuin and RNase B pulled down by Fbs1 beads in LS and HS conditions. Asterisk denotes a small amount of SNAP-Fbs1 that leaches from the Fbs1 beads. N-glycan structures present within fetuin and RNase B are illustrated. A representative SDS–PAGE gel is shown from two experiments. ( c ) Reciprocal pulldown of SNAP-Fbs1 by denatured fetuin or RNase B beads in LS or HS conditions. A representative SDS–PAGE gel is shown from two experiments. ( d ) HS conditions have no effect on Fbs1 binding to asialo-SGP-TMR. SGP-TMR was trimmed with α2-3,6,8 Neuraminidase to produce asialo-SGP-TMR (structures shown in Fig. 1d , glycopeptide 1 and 2). SGP-TMR and asialo-SGP-TMR were incubated with Fbs1 beads in LS buffer or HS buffer. SGP-TMR or asialo-SGP-TMR relative affinity to Fbs1 is indicated by the recovery percentage. Results represent the mean±s.e.m. of three replicates.

    Techniques Used: Binding Assay, Sequencing, Incubation, SDS Page

    9) Product Images from "Glycosylation of Human IgA Directly Inhibits Influenza A and Other Sialic-Acid-Binding Viruses"

    Article Title: Glycosylation of Human IgA Directly Inhibits Influenza A and Other Sialic-Acid-Binding Viruses

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2018.03.027

    Impact of Fc Glycosylation (A) Impact of Fc glycosylation on neutralizing activity. Comparison of the neutralizing activities of wild-type IgG1, wild-type IgA1, and IgA1 in which the glycosylation consensus sequences at position 263 or 459 have been removed. (B) Comparison of the complex glycoforms of IgG1 and IgA1 and the oligomannose-type glycoform of IgA1 expressed in 293S cells (IgA S). (C) Impact of neuraminidase treatment on the neutralization of rg-A/Chicken/Vietnam/C58/2004 (H5N3). The neutralizing activity of the indicated antibodies incubated either with α2–3 neuraminidase from Salmonella typhimurium LT2 (NA S) or α2–3,6,8,9 neuraminidase A from Arthrobacter ureafaciens (NA A) was compared to that of the mock-treated IgA1 and IgG1 molecules. (D) Impact of Fc glycosylation on hemagglutination inhibition. A constant amount of the indicated virus was incubated with titrated amounts of the indicated antibodies and added to chicken erythrocytes that were then allowed to sediment at room temperature. (E) Impact of prolonged incubation with virus. Virus and titrated amounts of the indicated isotype of mAb 3.1 were incubated for either 1 hr or overnight before residual infectivity was determined.
    Figure Legend Snippet: Impact of Fc Glycosylation (A) Impact of Fc glycosylation on neutralizing activity. Comparison of the neutralizing activities of wild-type IgG1, wild-type IgA1, and IgA1 in which the glycosylation consensus sequences at position 263 or 459 have been removed. (B) Comparison of the complex glycoforms of IgG1 and IgA1 and the oligomannose-type glycoform of IgA1 expressed in 293S cells (IgA S). (C) Impact of neuraminidase treatment on the neutralization of rg-A/Chicken/Vietnam/C58/2004 (H5N3). The neutralizing activity of the indicated antibodies incubated either with α2–3 neuraminidase from Salmonella typhimurium LT2 (NA S) or α2–3,6,8,9 neuraminidase A from Arthrobacter ureafaciens (NA A) was compared to that of the mock-treated IgA1 and IgG1 molecules. (D) Impact of Fc glycosylation on hemagglutination inhibition. A constant amount of the indicated virus was incubated with titrated amounts of the indicated antibodies and added to chicken erythrocytes that were then allowed to sediment at room temperature. (E) Impact of prolonged incubation with virus. Virus and titrated amounts of the indicated isotype of mAb 3.1 were incubated for either 1 hr or overnight before residual infectivity was determined.

    Techniques Used: Activity Assay, Neutralization, Incubation, HI Assay, Infection

    10) Product Images from "Changes in canine serum N-glycosylation as a result of infection with the heartworm parasite Dirofilaria immitis"

    Article Title: Changes in canine serum N-glycosylation as a result of infection with the heartworm parasite Dirofilaria immitis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-35038-7

    Longitudinal serum N-glycosylation profiles of D. immitis infection in dogs. ( a ) HILIC-UPLC profiles of enzymatically released and fluorescently labeled serum N-glycans from dog ID 116 (longitudinal set) from weeks 0, 21, 23, 25 and 27 post-infection with D. immitis . Rel. abund., relative abundance. The glycan structures of the two dominant peaks are annotated. ( b ) Heatmap of the changes in the abundance of serum N-glycan classes in D. immitis infection. Adjusted p-values were determined using linear mixed-effects models. Blue, increase; red, decrease. Glycan classes were identified and quantified by exoglycosidase digestion with α1-2,4,6 Fucosidase, β1-4 Galactosidase or α2-3,6,8 Neuraminidase. (Supplementary Fig. S3 ; Table S4 ).
    Figure Legend Snippet: Longitudinal serum N-glycosylation profiles of D. immitis infection in dogs. ( a ) HILIC-UPLC profiles of enzymatically released and fluorescently labeled serum N-glycans from dog ID 116 (longitudinal set) from weeks 0, 21, 23, 25 and 27 post-infection with D. immitis . Rel. abund., relative abundance. The glycan structures of the two dominant peaks are annotated. ( b ) Heatmap of the changes in the abundance of serum N-glycan classes in D. immitis infection. Adjusted p-values were determined using linear mixed-effects models. Blue, increase; red, decrease. Glycan classes were identified and quantified by exoglycosidase digestion with α1-2,4,6 Fucosidase, β1-4 Galactosidase or α2-3,6,8 Neuraminidase. (Supplementary Fig. S3 ; Table S4 ).

    Techniques Used: Infection, Hydrophilic Interaction Liquid Chromatography, Labeling

    Significant changes of serum N-glycosylation in dogs with a patent D. immitis infection. Glycan peaks/classes were tested for significance using linear mixed-effects models. p-values were adjusted based on Benjamini and Hochberg method. The analysis is based on 5 biological and 2 technical replicates per group (healthy and disease; patent set). ( a ) Volcano Plot comparing serum N-glycan peaks from dogs infected with D. immitis to a healthy control group. Illustrated is the log2 fold change in glycan abundance and the negative log2 of adjusted p-values. The horizontal dashed line represents the adjusted p-value cutoff (0.05). The points above the dashed line are glycan peaks that decrease (red) and increase (blue) significantly. See Supplementary Table S4 for quantification data. ( b ) Significant changes in all analyzed glycan classes in dogs with a patent D. immitis infection. Glycan classes were identified and quantified by exoglycosidase digestion with α1-2,4,6 Fucosidase, β1-4 Galactosidase or α2-3,6,8 Neuraminidase. See Supplementary Fig. S4 for visualization of the glycan classes. Bar graphs show mean + s.d. Agalactosylation, adj. p-value = 0.001; galactosylation, adj. p-value = 0.003; core fucosylation, adj. p-value = 0.001; sialylation, adj. p-value = 0.001.
    Figure Legend Snippet: Significant changes of serum N-glycosylation in dogs with a patent D. immitis infection. Glycan peaks/classes were tested for significance using linear mixed-effects models. p-values were adjusted based on Benjamini and Hochberg method. The analysis is based on 5 biological and 2 technical replicates per group (healthy and disease; patent set). ( a ) Volcano Plot comparing serum N-glycan peaks from dogs infected with D. immitis to a healthy control group. Illustrated is the log2 fold change in glycan abundance and the negative log2 of adjusted p-values. The horizontal dashed line represents the adjusted p-value cutoff (0.05). The points above the dashed line are glycan peaks that decrease (red) and increase (blue) significantly. See Supplementary Table S4 for quantification data. ( b ) Significant changes in all analyzed glycan classes in dogs with a patent D. immitis infection. Glycan classes were identified and quantified by exoglycosidase digestion with α1-2,4,6 Fucosidase, β1-4 Galactosidase or α2-3,6,8 Neuraminidase. See Supplementary Fig. S4 for visualization of the glycan classes. Bar graphs show mean + s.d. Agalactosylation, adj. p-value = 0.001; galactosylation, adj. p-value = 0.003; core fucosylation, adj. p-value = 0.001; sialylation, adj. p-value = 0.001.

    Techniques Used: Infection

    11) Product Images from "Vitamin D binding protein isoforms as candidate predictors of disease extension in childhood arthritis"

    Article Title: Vitamin D binding protein isoforms as candidate predictors of disease extension in childhood arthritis

    Journal: Journal of Proteomics

    doi: 10.1016/j.jprot.2012.06.024

    MALDI-TOF mass spectrometry of intact immunopurified VDBP and enzymatically released glycans diagnostic capacity. A. MALDI-TOF mass spectrometry of immunoprecipitated and linearized VDBP from a representative pool of study patients. The peak intensity represented in as an individual ‘densitometry’ lane below the chart, reflects higher plasma concentrations of VDBP. The majority of intact forms of VDBP from plasma and synovial fluid have a peak mass of 52,900 m / z (represented by the red dashed line). A peak shift towards the right resulting in a ‘shoulder’ is apparent, indicating higher mass variants or isoforms of VDBP are also present in both fluids. B. MALDI-TOF mass spectrometry of HILIC enriched glycan released by α2-3 neuraminidase digest from immunopurified VDBP from the synovial fluid of oligoarticular patients. Glycan peaks at 237.65 m / z and 435.58 m / z , representing release of sialic acid residues, are indicated by the red encircled bands within individual patient ‘densitometry’ lanes. There is a significant 72.4 fold difference in the normalized 237.65 m / z peak heights between persistent and extended-to-be oligoarticular patients, signifying few sialic acid modifications detected in the VDBP taken from the latter patients ( p = 0.0014). C. Receiver operator characteristic curves to test the sensitivity and specificity of VDBP (black line) and CRP (red line) ELISA concentration values and DIGE derived normalized volumes for spot 873 (green line) and sialic acid (peak 237.65 m / z ; blue line) released from VDBP to discern patients at risk of disease spread. Area under the curve and p values for each test are also included in the accompanying table.
    Figure Legend Snippet: MALDI-TOF mass spectrometry of intact immunopurified VDBP and enzymatically released glycans diagnostic capacity. A. MALDI-TOF mass spectrometry of immunoprecipitated and linearized VDBP from a representative pool of study patients. The peak intensity represented in as an individual ‘densitometry’ lane below the chart, reflects higher plasma concentrations of VDBP. The majority of intact forms of VDBP from plasma and synovial fluid have a peak mass of 52,900 m / z (represented by the red dashed line). A peak shift towards the right resulting in a ‘shoulder’ is apparent, indicating higher mass variants or isoforms of VDBP are also present in both fluids. B. MALDI-TOF mass spectrometry of HILIC enriched glycan released by α2-3 neuraminidase digest from immunopurified VDBP from the synovial fluid of oligoarticular patients. Glycan peaks at 237.65 m / z and 435.58 m / z , representing release of sialic acid residues, are indicated by the red encircled bands within individual patient ‘densitometry’ lanes. There is a significant 72.4 fold difference in the normalized 237.65 m / z peak heights between persistent and extended-to-be oligoarticular patients, signifying few sialic acid modifications detected in the VDBP taken from the latter patients ( p = 0.0014). C. Receiver operator characteristic curves to test the sensitivity and specificity of VDBP (black line) and CRP (red line) ELISA concentration values and DIGE derived normalized volumes for spot 873 (green line) and sialic acid (peak 237.65 m / z ; blue line) released from VDBP to discern patients at risk of disease spread. Area under the curve and p values for each test are also included in the accompanying table.

    Techniques Used: Mass Spectrometry, Diagnostic Assay, Immunoprecipitation, Hydrophilic Interaction Liquid Chromatography, Enzyme-linked Immunosorbent Assay, Concentration Assay, Derivative Assay

    12) Product Images from "An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides"

    Article Title: An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

    Journal: Nature Communications

    doi: 10.1038/ncomms15487

    High-salt conditions increase complex N-glycomolecule binding to wt Fbs1. ( a ) The presence of 2 M NaCl increases SGP-TMR binding to wt Fbs1 in an N-glycan-dependent manner. PNGase F (+) indicates SGP-TMR was pretreated with PNGase F to cleave the glycan from the fluorophore-labelled peptide (sequence KVANKT). SGP-TMR with or without PNGase F treatment was incubated with Fbs1 beads in low-salt (LS) conditions or high-salt (HS) conditions. SGP-TMR binding to Fbs1 beads was measured, and affinity to Fbs1 is indicated by percentage of recovery (amount of bound SGP-TMR/amount of input SGP-TMR). Results represent the mean±s.e.m. of three replicates. ( b ) HS conditions increase Fbs1 binding to sialylated fetuin relative to RNase B, which contains high-mannose N-glycans. A mixture of denatured fetuin and RNase B was subjected to an Fbs1 bead pulldown assay. Lane 1 indicates the input ratio of fetuin to RNase B. Lanes 2 and 3 show the amounts of fetuin and RNase B pulled down by Fbs1 beads in LS and HS conditions. Asterisk denotes a small amount of SNAP-Fbs1 that leaches from the Fbs1 beads. N-glycan structures present within fetuin and RNase B are illustrated. A representative SDS–PAGE gel is shown from two experiments. ( c ) Reciprocal pulldown of SNAP-Fbs1 by denatured fetuin or RNase B beads in LS or HS conditions. A representative SDS–PAGE gel is shown from two experiments. ( d ) HS conditions have no effect on Fbs1 binding to asialo-SGP-TMR. SGP-TMR was trimmed with α2-3,6,8 Neuraminidase to produce asialo-SGP-TMR (structures shown in Fig. 1d , glycopeptide 1 and 2). SGP-TMR and asialo-SGP-TMR were incubated with Fbs1 beads in LS buffer or HS buffer. SGP-TMR or asialo-SGP-TMR relative affinity to Fbs1 is indicated by the recovery percentage. Results represent the mean±s.e.m. of three replicates.
    Figure Legend Snippet: High-salt conditions increase complex N-glycomolecule binding to wt Fbs1. ( a ) The presence of 2 M NaCl increases SGP-TMR binding to wt Fbs1 in an N-glycan-dependent manner. PNGase F (+) indicates SGP-TMR was pretreated with PNGase F to cleave the glycan from the fluorophore-labelled peptide (sequence KVANKT). SGP-TMR with or without PNGase F treatment was incubated with Fbs1 beads in low-salt (LS) conditions or high-salt (HS) conditions. SGP-TMR binding to Fbs1 beads was measured, and affinity to Fbs1 is indicated by percentage of recovery (amount of bound SGP-TMR/amount of input SGP-TMR). Results represent the mean±s.e.m. of three replicates. ( b ) HS conditions increase Fbs1 binding to sialylated fetuin relative to RNase B, which contains high-mannose N-glycans. A mixture of denatured fetuin and RNase B was subjected to an Fbs1 bead pulldown assay. Lane 1 indicates the input ratio of fetuin to RNase B. Lanes 2 and 3 show the amounts of fetuin and RNase B pulled down by Fbs1 beads in LS and HS conditions. Asterisk denotes a small amount of SNAP-Fbs1 that leaches from the Fbs1 beads. N-glycan structures present within fetuin and RNase B are illustrated. A representative SDS–PAGE gel is shown from two experiments. ( c ) Reciprocal pulldown of SNAP-Fbs1 by denatured fetuin or RNase B beads in LS or HS conditions. A representative SDS–PAGE gel is shown from two experiments. ( d ) HS conditions have no effect on Fbs1 binding to asialo-SGP-TMR. SGP-TMR was trimmed with α2-3,6,8 Neuraminidase to produce asialo-SGP-TMR (structures shown in Fig. 1d , glycopeptide 1 and 2). SGP-TMR and asialo-SGP-TMR were incubated with Fbs1 beads in LS buffer or HS buffer. SGP-TMR or asialo-SGP-TMR relative affinity to Fbs1 is indicated by the recovery percentage. Results represent the mean±s.e.m. of three replicates.

    Techniques Used: Binding Assay, Sequencing, Incubation, SDS Page

    13) Product Images from "The human milk oligosaccharide disialyllacto-N-tetraose prevents necrotising enterocolitis in neonatal rats"

    Article Title: The human milk oligosaccharide disialyllacto-N-tetraose prevents necrotising enterocolitis in neonatal rats

    Journal: Gut

    doi: 10.1136/gutjnl-2011-301404

    The necrotising enterocolitis-protective human milk oligosaccharide (HMO) is disialyllacto-N-tetraose (DSLNT). (A) Linkage specific neuraminidase treatment shows the presence of one α2–3- and one α2–6-linked N-acetyl-neuraminic
    Figure Legend Snippet: The necrotising enterocolitis-protective human milk oligosaccharide (HMO) is disialyllacto-N-tetraose (DSLNT). (A) Linkage specific neuraminidase treatment shows the presence of one α2–3- and one α2–6-linked N-acetyl-neuraminic

    Techniques Used:

    14) Product Images from "Fucosyl-Agalactosyl IgG1 Induces Cholangiocarcinoma Metastasis and Early Recurrence by Activating Tumor-Associated Macrophage"

    Article Title: Fucosyl-Agalactosyl IgG1 Induces Cholangiocarcinoma Metastasis and Early Recurrence by Activating Tumor-Associated Macrophage

    Journal: Cancers

    doi: 10.3390/cancers10110460

    Induction of tumor-associated macrophage by agalactosylated IgG. ( A ) Numbers of CD163+ macrophages in the tumor foci of cholangiocarcinoma in patients with different tumor grades (left panel), with or without tumor metastasis (middle panel), or with different IgG 1 -G0F level (right panel) are shown in Tukey box-and-whisker plots. A P -value in the left panel is obtained from the Kruskal–Wallis test. P -values in the middle and right panels are obtained from Mann–Whitney U tests. ( B ) A schematic representation of the depletion of terminal sialic acid and galactose moieties on serum IgG using α2-3,6,8 neuraminidase and β1-4 galactosidase S, respectively. The proportion of each glycoform on normal or galactose-and-sialic acid-removed (asialyl-agalactosyl) IgG 1 and IgG 2 are shown. ( C ) Messenger RNA levels of the macrophage marker CD68 and tumor-associated macrophage markers CD163 and CD204 in U-937 cells after treatments with 10 ng/mL of phorbol 12-myristate 13-acetate for 2 days and 10 mg/mL of IgG (blue bar, mock; red bar, agalactosyl IgG; green bar, normal serum IgG) for another 3 or 6 days, are shown in bar graphs as means with standard deviations. Results are obtained from three independent experiments. p -values are obtained from one-way analysis of variance with Scheffé post hoc tests. Immunoblotting assays to detect protein levels of ( D ) CD68, CD163, and CD204 at day 6 post-treatment and ( E ) CD64 (FcγRI) and CD16 (FcγRIII) in macrophagic U-937 cells and human peripheral macrophages are shown. (* p
    Figure Legend Snippet: Induction of tumor-associated macrophage by agalactosylated IgG. ( A ) Numbers of CD163+ macrophages in the tumor foci of cholangiocarcinoma in patients with different tumor grades (left panel), with or without tumor metastasis (middle panel), or with different IgG 1 -G0F level (right panel) are shown in Tukey box-and-whisker plots. A P -value in the left panel is obtained from the Kruskal–Wallis test. P -values in the middle and right panels are obtained from Mann–Whitney U tests. ( B ) A schematic representation of the depletion of terminal sialic acid and galactose moieties on serum IgG using α2-3,6,8 neuraminidase and β1-4 galactosidase S, respectively. The proportion of each glycoform on normal or galactose-and-sialic acid-removed (asialyl-agalactosyl) IgG 1 and IgG 2 are shown. ( C ) Messenger RNA levels of the macrophage marker CD68 and tumor-associated macrophage markers CD163 and CD204 in U-937 cells after treatments with 10 ng/mL of phorbol 12-myristate 13-acetate for 2 days and 10 mg/mL of IgG (blue bar, mock; red bar, agalactosyl IgG; green bar, normal serum IgG) for another 3 or 6 days, are shown in bar graphs as means with standard deviations. Results are obtained from three independent experiments. p -values are obtained from one-way analysis of variance with Scheffé post hoc tests. Immunoblotting assays to detect protein levels of ( D ) CD68, CD163, and CD204 at day 6 post-treatment and ( E ) CD64 (FcγRI) and CD16 (FcγRIII) in macrophagic U-937 cells and human peripheral macrophages are shown. (* p

    Techniques Used: Whisker Assay, MANN-WHITNEY, Marker

    15) Product Images from "Alterations in expressed prostate secretion-urine PSA N-glycosylation discriminate prostate cancer from benign prostate hyperplasia"

    Article Title: Alterations in expressed prostate secretion-urine PSA N-glycosylation discriminate prostate cancer from benign prostate hyperplasia

    Journal: Oncotarget

    doi: 10.18632/oncotarget.20299

    Representative UPLC chromatogram profiles of QC N-glycan samples (A) 42 peaks were identified in the glycan profiles of QC samples. (B) Glycan profiles before and after digestion with α2-3,6,8,9 Neuraminidase to remove all the sialic acids residues.
    Figure Legend Snippet: Representative UPLC chromatogram profiles of QC N-glycan samples (A) 42 peaks were identified in the glycan profiles of QC samples. (B) Glycan profiles before and after digestion with α2-3,6,8,9 Neuraminidase to remove all the sialic acids residues.

    Techniques Used:

    16) Product Images from "Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1"

    Article Title: Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32534-8

    SPR analysis of bindings of lectin and SKM9-2 to glycosidase-treated SKMepmin3. Purified SKMepmin3 was incubated with α2-3 Neuraminidase S, Neuraminidase A, or Neuraminidase A + O -Glycosidase; and then was immobilized as a ligand on Ni 2+ -binding sensor chip NTA. The analytes were used at a concentration of 5 µg/mL. ( a ) ABA, ( b ) Jacalin, ( c ) rACG, ( d ) SKM9-2. Glycosidase-digested sample was not tested in the experiment with rACG.
    Figure Legend Snippet: SPR analysis of bindings of lectin and SKM9-2 to glycosidase-treated SKMepmin3. Purified SKMepmin3 was incubated with α2-3 Neuraminidase S, Neuraminidase A, or Neuraminidase A + O -Glycosidase; and then was immobilized as a ligand on Ni 2+ -binding sensor chip NTA. The analytes were used at a concentration of 5 µg/mL. ( a ) ABA, ( b ) Jacalin, ( c ) rACG, ( d ) SKM9-2. Glycosidase-digested sample was not tested in the experiment with rACG.

    Techniques Used: SPR Assay, Purification, Incubation, Binding Assay, Chromatin Immunoprecipitation, Concentration Assay

    17) Product Images from "Characterizing Protein Glycosylation through On-Chip Glycan Modification and Probing"

    Article Title: Characterizing Protein Glycosylation through On-Chip Glycan Modification and Probing

    Journal: Analytical chemistry

    doi: 10.1021/acs.analchem.6b02998

    Test case: distinguishing α2,3 from α2,6 sialic acid
    Figure Legend Snippet: Test case: distinguishing α2,3 from α2,6 sialic acid

    Techniques Used:

    18) Product Images from "Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1"

    Article Title: Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32534-8

    SPR analysis of bindings of lectin and SKM9-2 to glycosidase-treated SKMepmin3. Purified SKMepmin3 was incubated with α2-3 Neuraminidase S, Neuraminidase A, or Neuraminidase A + O -Glycosidase; and then was immobilized as a ligand on Ni 2+ -binding sensor chip NTA. The analytes were used at a concentration of 5 µg/mL. ( a ) ABA, ( b ) Jacalin, ( c ) rACG, ( d ) SKM9-2. Glycosidase-digested sample was not tested in the experiment with rACG.
    Figure Legend Snippet: SPR analysis of bindings of lectin and SKM9-2 to glycosidase-treated SKMepmin3. Purified SKMepmin3 was incubated with α2-3 Neuraminidase S, Neuraminidase A, or Neuraminidase A + O -Glycosidase; and then was immobilized as a ligand on Ni 2+ -binding sensor chip NTA. The analytes were used at a concentration of 5 µg/mL. ( a ) ABA, ( b ) Jacalin, ( c ) rACG, ( d ) SKM9-2. Glycosidase-digested sample was not tested in the experiment with rACG.

    Techniques Used: SPR Assay, Purification, Incubation, Binding Assay, Chromatin Immunoprecipitation, Concentration Assay

    19) Product Images from "Glycosylation of Human IgA Directly Inhibits Influenza A and Other Sialic-Acid-Binding Viruses"

    Article Title: Glycosylation of Human IgA Directly Inhibits Influenza A and Other Sialic-Acid-Binding Viruses

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2018.03.027

    Impact of Fc Glycosylation (A) Impact of Fc glycosylation on neutralizing activity. Comparison of the neutralizing activities of wild-type IgG1, wild-type IgA1, and IgA1 in which the glycosylation consensus sequences at position 263 or 459 have been removed. (B) Comparison of the complex glycoforms of IgG1 and IgA1 and the oligomannose-type glycoform of IgA1 expressed in 293S cells (IgA S). (C) Impact of neuraminidase treatment on the neutralization of rg-A/Chicken/Vietnam/C58/2004 (H5N3). The neutralizing activity of the indicated antibodies incubated either with α2–3 neuraminidase from Salmonella typhimurium LT2 (NA S) or α2–3,6,8,9 neuraminidase A from Arthrobacter ureafaciens (NA A) was compared to that of the mock-treated IgA1 and IgG1 molecules. (D) Impact of Fc glycosylation on hemagglutination inhibition. A constant amount of the indicated virus was incubated with titrated amounts of the indicated antibodies and added to chicken erythrocytes that were then allowed to sediment at room temperature. (E) Impact of prolonged incubation with virus. Virus and titrated amounts of the indicated isotype of mAb 3.1 were incubated for either 1 hr or overnight before residual infectivity was determined.
    Figure Legend Snippet: Impact of Fc Glycosylation (A) Impact of Fc glycosylation on neutralizing activity. Comparison of the neutralizing activities of wild-type IgG1, wild-type IgA1, and IgA1 in which the glycosylation consensus sequences at position 263 or 459 have been removed. (B) Comparison of the complex glycoforms of IgG1 and IgA1 and the oligomannose-type glycoform of IgA1 expressed in 293S cells (IgA S). (C) Impact of neuraminidase treatment on the neutralization of rg-A/Chicken/Vietnam/C58/2004 (H5N3). The neutralizing activity of the indicated antibodies incubated either with α2–3 neuraminidase from Salmonella typhimurium LT2 (NA S) or α2–3,6,8,9 neuraminidase A from Arthrobacter ureafaciens (NA A) was compared to that of the mock-treated IgA1 and IgG1 molecules. (D) Impact of Fc glycosylation on hemagglutination inhibition. A constant amount of the indicated virus was incubated with titrated amounts of the indicated antibodies and added to chicken erythrocytes that were then allowed to sediment at room temperature. (E) Impact of prolonged incubation with virus. Virus and titrated amounts of the indicated isotype of mAb 3.1 were incubated for either 1 hr or overnight before residual infectivity was determined.

    Techniques Used: Activity Assay, Neutralization, Incubation, HI Assay, Infection

    20) Product Images from "An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides"

    Article Title: An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

    Journal: Nature Communications

    doi: 10.1038/ncomms15487

    High-salt conditions increase complex N-glycomolecule binding to wt Fbs1. ( a ) The presence of 2 M NaCl increases SGP-TMR binding to wt Fbs1 in an N-glycan-dependent manner. PNGase F (+) indicates SGP-TMR was pretreated with PNGase F to cleave the glycan from the fluorophore-labelled peptide (sequence KVANKT). SGP-TMR with or without PNGase F treatment was incubated with Fbs1 beads in low-salt (LS) conditions or high-salt (HS) conditions. SGP-TMR binding to Fbs1 beads was measured, and affinity to Fbs1 is indicated by percentage of recovery (amount of bound SGP-TMR/amount of input SGP-TMR). Results represent the mean±s.e.m. of three replicates. ( b ) HS conditions increase Fbs1 binding to sialylated fetuin relative to RNase B, which contains high-mannose N-glycans. A mixture of denatured fetuin and RNase B was subjected to an Fbs1 bead pulldown assay. Lane 1 indicates the input ratio of fetuin to RNase B. Lanes 2 and 3 show the amounts of fetuin and RNase B pulled down by Fbs1 beads in LS and HS conditions. Asterisk denotes a small amount of SNAP-Fbs1 that leaches from the Fbs1 beads. N-glycan structures present within fetuin and RNase B are illustrated. A representative SDS–PAGE gel is shown from two experiments. ( c ) Reciprocal pulldown of SNAP-Fbs1 by denatured fetuin or RNase B beads in LS or HS conditions. A representative SDS–PAGE gel is shown from two experiments. ( d ) HS conditions have no effect on Fbs1 binding to asialo-SGP-TMR. SGP-TMR was trimmed with α2-3,6,8 Neuraminidase to produce asialo-SGP-TMR (structures shown in Fig. 1d , glycopeptide 1 and 2). SGP-TMR and asialo-SGP-TMR were incubated with Fbs1 beads in LS buffer or HS buffer. SGP-TMR or asialo-SGP-TMR relative affinity to Fbs1 is indicated by the recovery percentage. Results represent the mean±s.e.m. of three replicates.
    Figure Legend Snippet: High-salt conditions increase complex N-glycomolecule binding to wt Fbs1. ( a ) The presence of 2 M NaCl increases SGP-TMR binding to wt Fbs1 in an N-glycan-dependent manner. PNGase F (+) indicates SGP-TMR was pretreated with PNGase F to cleave the glycan from the fluorophore-labelled peptide (sequence KVANKT). SGP-TMR with or without PNGase F treatment was incubated with Fbs1 beads in low-salt (LS) conditions or high-salt (HS) conditions. SGP-TMR binding to Fbs1 beads was measured, and affinity to Fbs1 is indicated by percentage of recovery (amount of bound SGP-TMR/amount of input SGP-TMR). Results represent the mean±s.e.m. of three replicates. ( b ) HS conditions increase Fbs1 binding to sialylated fetuin relative to RNase B, which contains high-mannose N-glycans. A mixture of denatured fetuin and RNase B was subjected to an Fbs1 bead pulldown assay. Lane 1 indicates the input ratio of fetuin to RNase B. Lanes 2 and 3 show the amounts of fetuin and RNase B pulled down by Fbs1 beads in LS and HS conditions. Asterisk denotes a small amount of SNAP-Fbs1 that leaches from the Fbs1 beads. N-glycan structures present within fetuin and RNase B are illustrated. A representative SDS–PAGE gel is shown from two experiments. ( c ) Reciprocal pulldown of SNAP-Fbs1 by denatured fetuin or RNase B beads in LS or HS conditions. A representative SDS–PAGE gel is shown from two experiments. ( d ) HS conditions have no effect on Fbs1 binding to asialo-SGP-TMR. SGP-TMR was trimmed with α2-3,6,8 Neuraminidase to produce asialo-SGP-TMR (structures shown in Fig. 1d , glycopeptide 1 and 2). SGP-TMR and asialo-SGP-TMR were incubated with Fbs1 beads in LS buffer or HS buffer. SGP-TMR or asialo-SGP-TMR relative affinity to Fbs1 is indicated by the recovery percentage. Results represent the mean±s.e.m. of three replicates.

    Techniques Used: Binding Assay, Sequencing, Incubation, SDS Page

    21) Product Images from "Human mastadenovirus-B (HAdV-B)-specific E3-CR1β and E3-CR1γ glycoproteins interact with each other and localize at the plasma membrane of non-polarized airway epithelial cells"

    Article Title: Human mastadenovirus-B (HAdV-B)-specific E3-CR1β and E3-CR1γ glycoproteins interact with each other and localize at the plasma membrane of non-polarized airway epithelial cells

    Journal: Virology

    doi: 10.1016/j.virol.2020.04.005

    HAdV-3 E3-20.1K is N-glycosylated whereas E3-20.5K is N-and O-glycosylated. A549 cells were infected with the HAdV-3 N-tag wt virus at a MOI of 10 pfu/cell. At 48 hpi cell lysates were prepared and treated for 1 h with PNGase F, or α2-3,6,8,9 neuraminidase A, or O-glycosidase either individually or sequentially with α2-3,6,8,9 neuraminidase A and o-glycosidase or with PNGase F, α2-3,6,8,9 neuraminidase A, and o-glycosidase in the order mentioned. After treatment, the lysates were analyzed for the expression of VSV-G E3-20.1K, HA E3-20.5K, and GAPDH (loading control) by SDS-PAGE and WB. The blot is representative of three independent experiments.
    Figure Legend Snippet: HAdV-3 E3-20.1K is N-glycosylated whereas E3-20.5K is N-and O-glycosylated. A549 cells were infected with the HAdV-3 N-tag wt virus at a MOI of 10 pfu/cell. At 48 hpi cell lysates were prepared and treated for 1 h with PNGase F, or α2-3,6,8,9 neuraminidase A, or O-glycosidase either individually or sequentially with α2-3,6,8,9 neuraminidase A and o-glycosidase or with PNGase F, α2-3,6,8,9 neuraminidase A, and o-glycosidase in the order mentioned. After treatment, the lysates were analyzed for the expression of VSV-G E3-20.1K, HA E3-20.5K, and GAPDH (loading control) by SDS-PAGE and WB. The blot is representative of three independent experiments.

    Techniques Used: Infection, Expressing, SDS Page, Western Blot

    22) Product Images from "HIV-1 incorporation of host-cell-derived glycosphingolipid GM3 allows for capture by mature dendritic cells"

    Article Title: HIV-1 incorporation of host-cell-derived glycosphingolipid GM3 allows for capture by mature dendritic cells

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

    doi: 10.1073/pnas.1201104109

    Enrichment of HIV-1 particles with α2–3-linked gangliosides results in enhanced capture by mDCs. HIV Lai particles were derived from HeLa cells treated with MeOH (mock), exogenous GM3 (+GM3) ( A – C ), or exogenous GM1 (+GM1) ( D –
    Figure Legend Snippet: Enrichment of HIV-1 particles with α2–3-linked gangliosides results in enhanced capture by mDCs. HIV Lai particles were derived from HeLa cells treated with MeOH (mock), exogenous GM3 (+GM3) ( A – C ), or exogenous GM1 (+GM1) ( D –

    Techniques Used: Derivative Assay

    Gangliosides with α2–3 NeuNAc linkages are important for HIV-1 capture by mDCs. ( A ) Gag-eGFP VLPs were mock treated or treated with 0.5 units/μL α2–3, 2–6, 2–8 NA. ( B ) Gag-eGFP VLPs were derived
    Figure Legend Snippet: Gangliosides with α2–3 NeuNAc linkages are important for HIV-1 capture by mDCs. ( A ) Gag-eGFP VLPs were mock treated or treated with 0.5 units/μL α2–3, 2–6, 2–8 NA. ( B ) Gag-eGFP VLPs were derived

    Techniques Used: Derivative Assay

    Liposomes with α2–3-linked gangliosides are captured by mDCs and compete HIV-1 for binding. Lipid vesicles comprised 54% DPPC, 1% PS, 45% cholesterol, and a fluorescent tag (blank). Modified vesicles contained 1% of Cer, Gal, GM3 (α2–3
    Figure Legend Snippet: Liposomes with α2–3-linked gangliosides are captured by mDCs and compete HIV-1 for binding. Lipid vesicles comprised 54% DPPC, 1% PS, 45% cholesterol, and a fluorescent tag (blank). Modified vesicles contained 1% of Cer, Gal, GM3 (α2–3

    Techniques Used: Binding Assay, Modification

    23) Product Images from "Elucidation of a Human Urine Metabolite as a Seryl-Leucine Glycopeptide and as a Biomarker of Effective Anti-Tuberculosis Therapy"

    Article Title: Elucidation of a Human Urine Metabolite as a Seryl-Leucine Glycopeptide and as a Biomarker of Effective Anti-Tuberculosis Therapy

    Journal: ACS Infectious Diseases

    doi: 10.1021/acsinfecdis.8b00241

    Confirmation of seryl-leucine peptide and SLC1G structure. EIC for m / z 219.1328 in LC-MS spectra of α2-3,6,8 neuraminidase and O -glycosidase treated MF 874.3547 (A) and seryl-leucine standard (C). MS/MS of m / z 219.1328 from α2-3,6,8 neuraminidase and O -glycosidase treated MF 874.3547 (B) and the seryl-leucine standard (D). The confirmed structure of Neu5Acα2-3Galβ1-3GalNAcα1- O -SerLeu for MF 874.3547 (E).
    Figure Legend Snippet: Confirmation of seryl-leucine peptide and SLC1G structure. EIC for m / z 219.1328 in LC-MS spectra of α2-3,6,8 neuraminidase and O -glycosidase treated MF 874.3547 (A) and seryl-leucine standard (C). MS/MS of m / z 219.1328 from α2-3,6,8 neuraminidase and O -glycosidase treated MF 874.3547 (B) and the seryl-leucine standard (D). The confirmed structure of Neu5Acα2-3Galβ1-3GalNAcα1- O -SerLeu for MF 874.3547 (E).

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Enzymatic deglycosylation and MS confirmation of core 1 glycosylation. MF 874.3547 untreated (A, C, E, G, I) and treated with α2-3,6,8 neuraminidase and O -glycosidase (B, D, F, H, J) were analyzed by LC-MS, and the spectra were evaluated by extracted ion chromatogram (EIC) for intact glycopeptide (A and B), the glycopeptide minus Neu5Ac (C and D), the Hex–HexNAc disaccharide (E and F), Neu5Ac (G and H), and the deglycosylated putative diamino acid S/TX (I and J). The * in panel C indicates a low level of the glycopeptide minus Neu5Ac ( m / z 584.2661) present in the undigested sample. Note the level of this product was considerably higher following digestion (G). In source fragmentation of SLC1G yielded the m / z 584.2661 product (# in panel C) at the same retention time as the undigested glycopeptide.
    Figure Legend Snippet: Enzymatic deglycosylation and MS confirmation of core 1 glycosylation. MF 874.3547 untreated (A, C, E, G, I) and treated with α2-3,6,8 neuraminidase and O -glycosidase (B, D, F, H, J) were analyzed by LC-MS, and the spectra were evaluated by extracted ion chromatogram (EIC) for intact glycopeptide (A and B), the glycopeptide minus Neu5Ac (C and D), the Hex–HexNAc disaccharide (E and F), Neu5Ac (G and H), and the deglycosylated putative diamino acid S/TX (I and J). The * in panel C indicates a low level of the glycopeptide minus Neu5Ac ( m / z 584.2661) present in the undigested sample. Note the level of this product was considerably higher following digestion (G). In source fragmentation of SLC1G yielded the m / z 584.2661 product (# in panel C) at the same retention time as the undigested glycopeptide.

    Techniques Used: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

    24) Product Images from "Alterations of the Human Skin N- and O-Glycome in Basal Cell Carcinoma and Squamous Cell Carcinoma"

    Article Title: Alterations of the Human Skin N- and O-Glycome in Basal Cell Carcinoma and Squamous Cell Carcinoma

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2018.00070

    N -glycosylation changes in squamous cell carcinoma (SCC). (A) Supervised hierarchical cluster analysis of healthy and tumor skin. Rows display each of the 15 patient glycan data (T, tumor tissue; C, healthy control). Columns indicate the N -glycan ID. Five clusters can be observed: in healthy tissue N -glycans with α2-3 linked N -acetylneuraminic acid (NeuAc) appeared to be present in increased levels whereas α2-6-NeuAc and oligomannose N -glycan levels were higher in tumor tissue. (B) Statistical evaluation of sialylated and oligomannose N -glycans uncovered significant changes [ p ≤ 0.04, using a t -test, indicated by an asterisk (*)]. Oligomannose N -glycans were upregulated whereas α2-3 linked NeuAc carrying N -glycans were down regulated in SCC.
    Figure Legend Snippet: N -glycosylation changes in squamous cell carcinoma (SCC). (A) Supervised hierarchical cluster analysis of healthy and tumor skin. Rows display each of the 15 patient glycan data (T, tumor tissue; C, healthy control). Columns indicate the N -glycan ID. Five clusters can be observed: in healthy tissue N -glycans with α2-3 linked N -acetylneuraminic acid (NeuAc) appeared to be present in increased levels whereas α2-6-NeuAc and oligomannose N -glycan levels were higher in tumor tissue. (B) Statistical evaluation of sialylated and oligomannose N -glycans uncovered significant changes [ p ≤ 0.04, using a t -test, indicated by an asterisk (*)]. Oligomannose N -glycans were upregulated whereas α2-3 linked NeuAc carrying N -glycans were down regulated in SCC.

    Techniques Used:

    Healthy human skin N -glycome. (A) Bean diagram representing the 10 most abundant N -glycans determined from 14 patients. Green bars indicate individual data points, the black line represents the median, and the gray area depicts the data density. Columns indicate the glycan structures given by their glycan ID (Table S1 in Supplementary Material). (B) Relative N -glycan class abundances found in healthy skin biopsies. Sialylated and fucosylated structures were the major components representing the human skin N -glycome. (C) Relative abundances of different structure features found on sialylated N -glycans. Blue bars represent sialylated N -glycans with and without core fucose depending on their sialic acid linkage, showing that core fucosylation was a more abundant feature on N -glycans carrying one or two α2-3 linked N -acetylneuraminic acid (NeuAc) residues (if both, α2-3 and α2-6 linkages were present on one N- glycan, this N -glycan was considered in both linkage categories). Overall, α2-6-linked NeuAc was a slightly more abundant feature compared with α2-3-linked NeuAc. Most of the sialylated N -glycans carried two NeuAc residues. Tri-antennary species were below 3% and rather low abundant using porous graphitized carbon, while multiplexed capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF) detected slightly higher levels of tri- and tetra-antennary N -glycans (see Supplementary file “xCGE-LIF quant.xlsx” in Supplementary Material).
    Figure Legend Snippet: Healthy human skin N -glycome. (A) Bean diagram representing the 10 most abundant N -glycans determined from 14 patients. Green bars indicate individual data points, the black line represents the median, and the gray area depicts the data density. Columns indicate the glycan structures given by their glycan ID (Table S1 in Supplementary Material). (B) Relative N -glycan class abundances found in healthy skin biopsies. Sialylated and fucosylated structures were the major components representing the human skin N -glycome. (C) Relative abundances of different structure features found on sialylated N -glycans. Blue bars represent sialylated N -glycans with and without core fucose depending on their sialic acid linkage, showing that core fucosylation was a more abundant feature on N -glycans carrying one or two α2-3 linked N -acetylneuraminic acid (NeuAc) residues (if both, α2-3 and α2-6 linkages were present on one N- glycan, this N -glycan was considered in both linkage categories). Overall, α2-6-linked NeuAc was a slightly more abundant feature compared with α2-3-linked NeuAc. Most of the sialylated N -glycans carried two NeuAc residues. Tri-antennary species were below 3% and rather low abundant using porous graphitized carbon, while multiplexed capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF) detected slightly higher levels of tri- and tetra-antennary N -glycans (see Supplementary file “xCGE-LIF quant.xlsx” in Supplementary Material).

    Techniques Used: Nucleic Acid Electrophoresis, Fluorescence

    25) Product Images from "HIV-1 incorporation of host-cell-derived glycosphingolipid GM3 allows for capture by mature dendritic cells"

    Article Title: HIV-1 incorporation of host-cell-derived glycosphingolipid GM3 allows for capture by mature dendritic cells

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

    doi: 10.1073/pnas.1201104109

    Enrichment of HIV-1 particles with α2–3-linked gangliosides results in enhanced capture by mDCs. HIV Lai particles were derived from HeLa cells treated with MeOH (mock), exogenous GM3 (+GM3) ( A – C ), or exogenous GM1 (+GM1) ( D –
    Figure Legend Snippet: Enrichment of HIV-1 particles with α2–3-linked gangliosides results in enhanced capture by mDCs. HIV Lai particles were derived from HeLa cells treated with MeOH (mock), exogenous GM3 (+GM3) ( A – C ), or exogenous GM1 (+GM1) ( D –

    Techniques Used: Derivative Assay

    Gangliosides with α2–3 NeuNAc linkages are important for HIV-1 capture by mDCs. ( A ) Gag-eGFP VLPs were mock treated or treated with 0.5 units/μL α2–3, 2–6, 2–8 NA. ( B ) Gag-eGFP VLPs were derived
    Figure Legend Snippet: Gangliosides with α2–3 NeuNAc linkages are important for HIV-1 capture by mDCs. ( A ) Gag-eGFP VLPs were mock treated or treated with 0.5 units/μL α2–3, 2–6, 2–8 NA. ( B ) Gag-eGFP VLPs were derived

    Techniques Used: Derivative Assay

    Liposomes with α2–3-linked gangliosides are captured by mDCs and compete HIV-1 for binding. Lipid vesicles comprised 54% DPPC, 1% PS, 45% cholesterol, and a fluorescent tag (blank). Modified vesicles contained 1% of Cer, Gal, GM3 (α2–3
    Figure Legend Snippet: Liposomes with α2–3-linked gangliosides are captured by mDCs and compete HIV-1 for binding. Lipid vesicles comprised 54% DPPC, 1% PS, 45% cholesterol, and a fluorescent tag (blank). Modified vesicles contained 1% of Cer, Gal, GM3 (α2–3

    Techniques Used: Binding Assay, Modification

    26) Product Images from "An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides"

    Article Title: An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

    Journal: Nature Communications

    doi: 10.1038/ncomms15487

    High-salt conditions increase complex N-glycomolecule binding to wt Fbs1. ( a ) The presence of 2 M NaCl increases SGP-TMR binding to wt Fbs1 in an N-glycan-dependent manner. PNGase F (+) indicates SGP-TMR was pretreated with PNGase F to cleave the glycan from the fluorophore-labelled peptide (sequence KVANKT). SGP-TMR with or without PNGase F treatment was incubated with Fbs1 beads in low-salt (LS) conditions or high-salt (HS) conditions. SGP-TMR binding to Fbs1 beads was measured, and affinity to Fbs1 is indicated by percentage of recovery (amount of bound SGP-TMR/amount of input SGP-TMR). Results represent the mean±s.e.m. of three replicates. ( b ) HS conditions increase Fbs1 binding to sialylated fetuin relative to RNase B, which contains high-mannose N-glycans. A mixture of denatured fetuin and RNase B was subjected to an Fbs1 bead pulldown assay. Lane 1 indicates the input ratio of fetuin to RNase B. Lanes 2 and 3 show the amounts of fetuin and RNase B pulled down by Fbs1 beads in LS and HS conditions. Asterisk denotes a small amount of SNAP-Fbs1 that leaches from the Fbs1 beads. N-glycan structures present within fetuin and RNase B are illustrated. A representative SDS–PAGE gel is shown from two experiments. ( c ) Reciprocal pulldown of SNAP-Fbs1 by denatured fetuin or RNase B beads in LS or HS conditions. A representative SDS–PAGE gel is shown from two experiments. ( d ) HS conditions have no effect on Fbs1 binding to asialo-SGP-TMR. SGP-TMR was trimmed with α2-3,6,8 Neuraminidase to produce asialo-SGP-TMR (structures shown in Fig. 1d , glycopeptide 1 and 2). SGP-TMR and asialo-SGP-TMR were incubated with Fbs1 beads in LS buffer or HS buffer. SGP-TMR or asialo-SGP-TMR relative affinity to Fbs1 is indicated by the recovery percentage. Results represent the mean±s.e.m. of three replicates.
    Figure Legend Snippet: High-salt conditions increase complex N-glycomolecule binding to wt Fbs1. ( a ) The presence of 2 M NaCl increases SGP-TMR binding to wt Fbs1 in an N-glycan-dependent manner. PNGase F (+) indicates SGP-TMR was pretreated with PNGase F to cleave the glycan from the fluorophore-labelled peptide (sequence KVANKT). SGP-TMR with or without PNGase F treatment was incubated with Fbs1 beads in low-salt (LS) conditions or high-salt (HS) conditions. SGP-TMR binding to Fbs1 beads was measured, and affinity to Fbs1 is indicated by percentage of recovery (amount of bound SGP-TMR/amount of input SGP-TMR). Results represent the mean±s.e.m. of three replicates. ( b ) HS conditions increase Fbs1 binding to sialylated fetuin relative to RNase B, which contains high-mannose N-glycans. A mixture of denatured fetuin and RNase B was subjected to an Fbs1 bead pulldown assay. Lane 1 indicates the input ratio of fetuin to RNase B. Lanes 2 and 3 show the amounts of fetuin and RNase B pulled down by Fbs1 beads in LS and HS conditions. Asterisk denotes a small amount of SNAP-Fbs1 that leaches from the Fbs1 beads. N-glycan structures present within fetuin and RNase B are illustrated. A representative SDS–PAGE gel is shown from two experiments. ( c ) Reciprocal pulldown of SNAP-Fbs1 by denatured fetuin or RNase B beads in LS or HS conditions. A representative SDS–PAGE gel is shown from two experiments. ( d ) HS conditions have no effect on Fbs1 binding to asialo-SGP-TMR. SGP-TMR was trimmed with α2-3,6,8 Neuraminidase to produce asialo-SGP-TMR (structures shown in Fig. 1d , glycopeptide 1 and 2). SGP-TMR and asialo-SGP-TMR were incubated with Fbs1 beads in LS buffer or HS buffer. SGP-TMR or asialo-SGP-TMR relative affinity to Fbs1 is indicated by the recovery percentage. Results represent the mean±s.e.m. of three replicates.

    Techniques Used: Binding Assay, Sequencing, Incubation, SDS Page

    27) Product Images from "Characterizing Protein Glycosylation through On-Chip Glycan Modification and Probing"

    Article Title: Characterizing Protein Glycosylation through On-Chip Glycan Modification and Probing

    Journal: Analytical chemistry

    doi: 10.1021/acs.analchem.6b02998

    Test case: distinguishing α2,3 from α2,6 sialic acid
    Figure Legend Snippet: Test case: distinguishing α2,3 from α2,6 sialic acid

    Techniques Used:

    Related Articles

    Modification:

    Article Title: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo
    Article Snippet: .. Removal of sialic acid modification on NCAM1Soluble or membrane fraction of HC and OB from BACE1+/+ and BACE1−/− mice was incubated with or without the α2-3,6,8,9 Neuraminidase A (Sialidase; P0722; New England Biolabs) in a reaction solution, which is provided by the manufacturer, to remove the sialic acid from NCAM1 proteins at 37 °C overnight. ..

    Mouse Assay:

    Article Title: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo
    Article Snippet: .. Removal of sialic acid modification on NCAM1Soluble or membrane fraction of HC and OB from BACE1+/+ and BACE1−/− mice was incubated with or without the α2-3,6,8,9 Neuraminidase A (Sialidase; P0722; New England Biolabs) in a reaction solution, which is provided by the manufacturer, to remove the sialic acid from NCAM1 proteins at 37 °C overnight. ..

    Incubation:

    Article Title: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo
    Article Snippet: .. Removal of sialic acid modification on NCAM1Soluble or membrane fraction of HC and OB from BACE1+/+ and BACE1−/− mice was incubated with or without the α2-3,6,8,9 Neuraminidase A (Sialidase; P0722; New England Biolabs) in a reaction solution, which is provided by the manufacturer, to remove the sialic acid from NCAM1 proteins at 37 °C overnight. ..

    Article Title: Detection of Chemotherapy-Resistant Pancreatic Cancer Using a Glycan Biomarker, sTRA
    Article Snippet: .. After sample incubation we prepared α2-3 neuraminidase (P0728L, New England Biolabs, Ipswich, MA) at a concentration of 250 U/mL in the supplied reaction buffer and incubated on arrays overnight at 37 °C. ..

    Article Title: The Small t Antigen of JC Virus Antagonizes RIG-I-Mediated Innate Immunity by Inhibiting TRIM25’s RNA Binding Ability
    Article Snippet: .. Twenty-five units of Benzonase nuclease (Sigma)/ml and 20 U/ml of α2-3,6,8 neuraminidase (New England BioLabs) were added to the cell suspension and incubated overnight in a water bath at 37°C. ..

    Article Title: Measuring Site-specific Glycosylation Similarity between Influenza a Virus Variants with Statistical Certainty
    Article Snippet: .. Before HILIC enrichment, the digested AGP glycopeptides were incubated with 200 units of α2-3,6,8 Neuraminidase (New England Biolabs # P0720S) for every 20 μg of glycoprotein overnight at 37°C. ..

    Article Title: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo
    Article Snippet: .. After washing the beads with PBS, the beads were incubated with or without the α2-3,6,8,9 Neuraminidase A (Sialidase; P0722; New England Biolabs) in a reaction solution, which is provided by the manufacturer, to remove sialic acid from purified NCAM1 proteins at 37 °C overnight. ..

    Concentration Assay:

    Article Title: Detection of Chemotherapy-Resistant Pancreatic Cancer Using a Glycan Biomarker, sTRA
    Article Snippet: .. After sample incubation we prepared α2-3 neuraminidase (P0728L, New England Biolabs, Ipswich, MA) at a concentration of 250 U/mL in the supplied reaction buffer and incubated on arrays overnight at 37 °C. ..

    Hydrophilic Interaction Liquid Chromatography:

    Article Title: Measuring Site-specific Glycosylation Similarity between Influenza a Virus Variants with Statistical Certainty
    Article Snippet: .. Before HILIC enrichment, the digested AGP glycopeptides were incubated with 200 units of α2-3,6,8 Neuraminidase (New England Biolabs # P0720S) for every 20 μg of glycoprotein overnight at 37°C. ..

    Purification:

    Article Title: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo
    Article Snippet: .. After washing the beads with PBS, the beads were incubated with or without the α2-3,6,8,9 Neuraminidase A (Sialidase; P0722; New England Biolabs) in a reaction solution, which is provided by the manufacturer, to remove sialic acid from purified NCAM1 proteins at 37 °C overnight. ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    New England Biolabs α2 3 neuraminidase
    EICs of A2G2S2, A2G2S1 and A2G2 glycopeptides in non-treated (a-c) , <t>α2–3</t> neuraminidase treated (d-f) and α2–3,6,8,9 neuraminidase treated samples (g-i) . The peaks at 37.3 min and 39.2 min in (a) showed sensitivity for α2–3 neuraminidase, which disappeared after enzymatic digestion (d) , indicating these two peaks were containing at least one sialic acid that were α2–3 linked to galactose. Upon α2–3 neuraminidase digestion, A2G2S1 can be detected (e) , which were not originally in sample (b) . Since the 35.5 min peak in (a) remained after α2–3 neuraminidase digestion (d) , both the sialic acid linkages can be assigned α2–6. Comparing the intensities between (e) and (f) , the 37.3 min peak in (a) .
    α2 3 Neuraminidase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α2 3 neuraminidase/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    α2 3 neuraminidase - by Bioz Stars, 2021-09
    95/100 stars
      Buy from Supplier

    Image Search Results


    EICs of A2G2S2, A2G2S1 and A2G2 glycopeptides in non-treated (a-c) , α2–3 neuraminidase treated (d-f) and α2–3,6,8,9 neuraminidase treated samples (g-i) . The peaks at 37.3 min and 39.2 min in (a) showed sensitivity for α2–3 neuraminidase, which disappeared after enzymatic digestion (d) , indicating these two peaks were containing at least one sialic acid that were α2–3 linked to galactose. Upon α2–3 neuraminidase digestion, A2G2S1 can be detected (e) , which were not originally in sample (b) . Since the 35.5 min peak in (a) remained after α2–3 neuraminidase digestion (d) , both the sialic acid linkages can be assigned α2–6. Comparing the intensities between (e) and (f) , the 37.3 min peak in (a) .

    Journal: Analytical chemistry

    Article Title: Isomeric Separation of N-Glycopeptides Derived from Glycoproteins by Porous Graphitic Carbon (PGC) LC-MS/MS

    doi: 10.1021/acs.analchem.0c00668

    Figure Lengend Snippet: EICs of A2G2S2, A2G2S1 and A2G2 glycopeptides in non-treated (a-c) , α2–3 neuraminidase treated (d-f) and α2–3,6,8,9 neuraminidase treated samples (g-i) . The peaks at 37.3 min and 39.2 min in (a) showed sensitivity for α2–3 neuraminidase, which disappeared after enzymatic digestion (d) , indicating these two peaks were containing at least one sialic acid that were α2–3 linked to galactose. Upon α2–3 neuraminidase digestion, A2G2S1 can be detected (e) , which were not originally in sample (b) . Since the 35.5 min peak in (a) remained after α2–3 neuraminidase digestion (d) , both the sialic acid linkages can be assigned α2–6. Comparing the intensities between (e) and (f) , the 37.3 min peak in (a) .

    Article Snippet: Murine IgG1 (Intact mAb Mass Check Standard) was obtained from Waters Corporation (Milford, MA). α2–3 neuraminidase, α2–3,6,8,9 neuraminidase, β1–3 galactosidase, β1–4 galactosidase and β1–3,4 galactosidase were acquired from New England Biolabs (Ipswich, MA).

    Techniques:

    N -glycosylation changes in squamous cell carcinoma (SCC). (A) Supervised hierarchical cluster analysis of healthy and tumor skin. Rows display each of the 15 patient glycan data (T, tumor tissue; C, healthy control). Columns indicate the N -glycan ID. Five clusters can be observed: in healthy tissue N -glycans with α2-3 linked N -acetylneuraminic acid (NeuAc) appeared to be present in increased levels whereas α2-6-NeuAc and oligomannose N -glycan levels were higher in tumor tissue. (B) Statistical evaluation of sialylated and oligomannose N -glycans uncovered significant changes [ p ≤ 0.04, using a t -test, indicated by an asterisk (*)]. Oligomannose N -glycans were upregulated whereas α2-3 linked NeuAc carrying N -glycans were down regulated in SCC.

    Journal: Frontiers in Oncology

    Article Title: Alterations of the Human Skin N- and O-Glycome in Basal Cell Carcinoma and Squamous Cell Carcinoma

    doi: 10.3389/fonc.2018.00070

    Figure Lengend Snippet: N -glycosylation changes in squamous cell carcinoma (SCC). (A) Supervised hierarchical cluster analysis of healthy and tumor skin. Rows display each of the 15 patient glycan data (T, tumor tissue; C, healthy control). Columns indicate the N -glycan ID. Five clusters can be observed: in healthy tissue N -glycans with α2-3 linked N -acetylneuraminic acid (NeuAc) appeared to be present in increased levels whereas α2-6-NeuAc and oligomannose N -glycan levels were higher in tumor tissue. (B) Statistical evaluation of sialylated and oligomannose N -glycans uncovered significant changes [ p ≤ 0.04, using a t -test, indicated by an asterisk (*)]. Oligomannose N -glycans were upregulated whereas α2-3 linked NeuAc carrying N -glycans were down regulated in SCC.

    Article Snippet: The glycan sample was reconstituted in 10 μL 25 mM ammonium acetate buffer (pH 5.5) and 25 U of a α2-3-specific neuraminidase (NEB, Ipswitch, MA, USA) were added.

    Techniques:

    Healthy human skin N -glycome. (A) Bean diagram representing the 10 most abundant N -glycans determined from 14 patients. Green bars indicate individual data points, the black line represents the median, and the gray area depicts the data density. Columns indicate the glycan structures given by their glycan ID (Table S1 in Supplementary Material). (B) Relative N -glycan class abundances found in healthy skin biopsies. Sialylated and fucosylated structures were the major components representing the human skin N -glycome. (C) Relative abundances of different structure features found on sialylated N -glycans. Blue bars represent sialylated N -glycans with and without core fucose depending on their sialic acid linkage, showing that core fucosylation was a more abundant feature on N -glycans carrying one or two α2-3 linked N -acetylneuraminic acid (NeuAc) residues (if both, α2-3 and α2-6 linkages were present on one N- glycan, this N -glycan was considered in both linkage categories). Overall, α2-6-linked NeuAc was a slightly more abundant feature compared with α2-3-linked NeuAc. Most of the sialylated N -glycans carried two NeuAc residues. Tri-antennary species were below 3% and rather low abundant using porous graphitized carbon, while multiplexed capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF) detected slightly higher levels of tri- and tetra-antennary N -glycans (see Supplementary file “xCGE-LIF quant.xlsx” in Supplementary Material).

    Journal: Frontiers in Oncology

    Article Title: Alterations of the Human Skin N- and O-Glycome in Basal Cell Carcinoma and Squamous Cell Carcinoma

    doi: 10.3389/fonc.2018.00070

    Figure Lengend Snippet: Healthy human skin N -glycome. (A) Bean diagram representing the 10 most abundant N -glycans determined from 14 patients. Green bars indicate individual data points, the black line represents the median, and the gray area depicts the data density. Columns indicate the glycan structures given by their glycan ID (Table S1 in Supplementary Material). (B) Relative N -glycan class abundances found in healthy skin biopsies. Sialylated and fucosylated structures were the major components representing the human skin N -glycome. (C) Relative abundances of different structure features found on sialylated N -glycans. Blue bars represent sialylated N -glycans with and without core fucose depending on their sialic acid linkage, showing that core fucosylation was a more abundant feature on N -glycans carrying one or two α2-3 linked N -acetylneuraminic acid (NeuAc) residues (if both, α2-3 and α2-6 linkages were present on one N- glycan, this N -glycan was considered in both linkage categories). Overall, α2-6-linked NeuAc was a slightly more abundant feature compared with α2-3-linked NeuAc. Most of the sialylated N -glycans carried two NeuAc residues. Tri-antennary species were below 3% and rather low abundant using porous graphitized carbon, while multiplexed capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF) detected slightly higher levels of tri- and tetra-antennary N -glycans (see Supplementary file “xCGE-LIF quant.xlsx” in Supplementary Material).

    Article Snippet: The glycan sample was reconstituted in 10 μL 25 mM ammonium acetate buffer (pH 5.5) and 25 U of a α2-3-specific neuraminidase (NEB, Ipswitch, MA, USA) were added.

    Techniques: Nucleic Acid Electrophoresis, Fluorescence

    Impact of Fc Glycosylation (A) Impact of Fc glycosylation on neutralizing activity. Comparison of the neutralizing activities of wild-type IgG1, wild-type IgA1, and IgA1 in which the glycosylation consensus sequences at position 263 or 459 have been removed. (B) Comparison of the complex glycoforms of IgG1 and IgA1 and the oligomannose-type glycoform of IgA1 expressed in 293S cells (IgA S). (C) Impact of neuraminidase treatment on the neutralization of rg-A/Chicken/Vietnam/C58/2004 (H5N3). The neutralizing activity of the indicated antibodies incubated either with α2–3 neuraminidase from Salmonella typhimurium LT2 (NA S) or α2–3,6,8,9 neuraminidase A from Arthrobacter ureafaciens (NA A) was compared to that of the mock-treated IgA1 and IgG1 molecules. (D) Impact of Fc glycosylation on hemagglutination inhibition. A constant amount of the indicated virus was incubated with titrated amounts of the indicated antibodies and added to chicken erythrocytes that were then allowed to sediment at room temperature. (E) Impact of prolonged incubation with virus. Virus and titrated amounts of the indicated isotype of mAb 3.1 were incubated for either 1 hr or overnight before residual infectivity was determined.

    Journal: Cell Reports

    Article Title: Glycosylation of Human IgA Directly Inhibits Influenza A and Other Sialic-Acid-Binding Viruses

    doi: 10.1016/j.celrep.2018.03.027

    Figure Lengend Snippet: Impact of Fc Glycosylation (A) Impact of Fc glycosylation on neutralizing activity. Comparison of the neutralizing activities of wild-type IgG1, wild-type IgA1, and IgA1 in which the glycosylation consensus sequences at position 263 or 459 have been removed. (B) Comparison of the complex glycoforms of IgG1 and IgA1 and the oligomannose-type glycoform of IgA1 expressed in 293S cells (IgA S). (C) Impact of neuraminidase treatment on the neutralization of rg-A/Chicken/Vietnam/C58/2004 (H5N3). The neutralizing activity of the indicated antibodies incubated either with α2–3 neuraminidase from Salmonella typhimurium LT2 (NA S) or α2–3,6,8,9 neuraminidase A from Arthrobacter ureafaciens (NA A) was compared to that of the mock-treated IgA1 and IgG1 molecules. (D) Impact of Fc glycosylation on hemagglutination inhibition. A constant amount of the indicated virus was incubated with titrated amounts of the indicated antibodies and added to chicken erythrocytes that were then allowed to sediment at room temperature. (E) Impact of prolonged incubation with virus. Virus and titrated amounts of the indicated isotype of mAb 3.1 were incubated for either 1 hr or overnight before residual infectivity was determined.

    Article Snippet: The 2-AA labeled glycans were sequentially digested using the following exoglycosidases according to the manufacturers’ instructions: α2-3,6,8 neuraminidase from Clostridium perfringens (New England Biolabs, Hertfordshire, UK), α2-3 neuraminidase from Streptococcus pneumoniae (New England BioLabs, Hertfordshire, UK), β1,4-galactosidase from Bacteroides fragilis (New England Biolabs, Hertfordshire, UK), and α-L-fucosidase from bovine kidney (Sigma-Aldrich, Dorset, UK), β- N -acetylglucosaminidase from Xanthomonas manihotis (New England Biolabs, Hertfordshire, UK), and α(1–2,3,6)-mannosidase from Jack bean (Sigma-Aldrich, Dorset, UK).

    Techniques: Activity Assay, Neutralization, Incubation, HI Assay, Infection

    SPR analysis of bindings of lectin and SKM9-2 to glycosidase-treated SKMepmin3. Purified SKMepmin3 was incubated with α2-3 Neuraminidase S, Neuraminidase A, or Neuraminidase A + O -Glycosidase; and then was immobilized as a ligand on Ni 2+ -binding sensor chip NTA. The analytes were used at a concentration of 5 µg/mL. ( a ) ABA, ( b ) Jacalin, ( c ) rACG, ( d ) SKM9-2. Glycosidase-digested sample was not tested in the experiment with rACG.

    Journal: Scientific Reports

    Article Title: Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1

    doi: 10.1038/s41598-018-32534-8

    Figure Lengend Snippet: SPR analysis of bindings of lectin and SKM9-2 to glycosidase-treated SKMepmin3. Purified SKMepmin3 was incubated with α2-3 Neuraminidase S, Neuraminidase A, or Neuraminidase A + O -Glycosidase; and then was immobilized as a ligand on Ni 2+ -binding sensor chip NTA. The analytes were used at a concentration of 5 µg/mL. ( a ) ABA, ( b ) Jacalin, ( c ) rACG, ( d ) SKM9-2. Glycosidase-digested sample was not tested in the experiment with rACG.

    Article Snippet: Deglycosylation analysis Cell lysate or SKMepmin3 was treated with α2-3 Neuraminidase S, α2-3, 6, 8, 9 Neuraminidase A, and O -Glycosidase (New England Biolabs Japan, Tokyo, Japan) in 50 mM sodium phosphate (pH 7.5) containing 1% Nonidet P-40, according to the manufacturer’s instructions.

    Techniques: SPR Assay, Purification, Incubation, Binding Assay, Chromatin Immunoprecipitation, Concentration Assay

    SKM9-2 epitope produced from mesothelioma cells. ( a ) Chromatogram of SKMepmin3 separation using anion exchange column. SKMepmin3 produced from HEK293T (blue line) or ACC-MESO4 (pink line) was affinity-purified using SKM9-2-immobilized resins and separated by Mini Q 4.6/50 PE. A major peak of SKMepmin3 purified from HEK293T contained two disialyl T antigens (Fig. 6 ). ( b ) Western blotting for epitope-fused GPI-anchor protein (7.6231) treated with neuraminidases. Cell lysate of 7.6231-transfected ACC-MESO1 (top panel), ACC-MESO4 (middle panel), or HEK293T (lower panel) was treated with α2-3 Neuraminidase S or Neuraminidase A, resolved by 4–15% SDS-PAGE, and analyzed by western blotting using SKM9-2. ( c ) Western blotting for full-length HEG1 treated with neuraminidases. Cell lysates of ACC-MESO4 (upper panel) and HEG1-transfected HEK293T (lower panel) were treated with α2-3 Neuraminidase S or Neuraminidase A and analyzed by 6% SDS-PAGE and western blotting using SKM9-2. ( d ) Schematic of SKM9-2 epitope. Full-length blots are presented in Supplementary Fig. S10 .

    Journal: Scientific Reports

    Article Title: Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1

    doi: 10.1038/s41598-018-32534-8

    Figure Lengend Snippet: SKM9-2 epitope produced from mesothelioma cells. ( a ) Chromatogram of SKMepmin3 separation using anion exchange column. SKMepmin3 produced from HEK293T (blue line) or ACC-MESO4 (pink line) was affinity-purified using SKM9-2-immobilized resins and separated by Mini Q 4.6/50 PE. A major peak of SKMepmin3 purified from HEK293T contained two disialyl T antigens (Fig. 6 ). ( b ) Western blotting for epitope-fused GPI-anchor protein (7.6231) treated with neuraminidases. Cell lysate of 7.6231-transfected ACC-MESO1 (top panel), ACC-MESO4 (middle panel), or HEK293T (lower panel) was treated with α2-3 Neuraminidase S or Neuraminidase A, resolved by 4–15% SDS-PAGE, and analyzed by western blotting using SKM9-2. ( c ) Western blotting for full-length HEG1 treated with neuraminidases. Cell lysates of ACC-MESO4 (upper panel) and HEG1-transfected HEK293T (lower panel) were treated with α2-3 Neuraminidase S or Neuraminidase A and analyzed by 6% SDS-PAGE and western blotting using SKM9-2. ( d ) Schematic of SKM9-2 epitope. Full-length blots are presented in Supplementary Fig. S10 .

    Article Snippet: Deglycosylation analysis Cell lysate or SKMepmin3 was treated with α2-3 Neuraminidase S, α2-3, 6, 8, 9 Neuraminidase A, and O -Glycosidase (New England Biolabs Japan, Tokyo, Japan) in 50 mM sodium phosphate (pH 7.5) containing 1% Nonidet P-40, according to the manufacturer’s instructions.

    Techniques: Produced, Affinity Purification, Purification, Western Blot, Transfection, SDS Page