fetuin  (New England Biolabs)


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    Name:
    Fetuin
    Description:
    Fetuin 500 ug
    Catalog Number:
    p6042s
    Price:
    74
    Size:
    500 ug
    Category:
    Glycoproteins
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    Structured Review

    New England Biolabs fetuin
    Fetuin
    Fetuin 500 ug
    https://www.bioz.com/result/fetuin/product/New England Biolabs
    Average 94 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    fetuin - by Bioz Stars, 2020-07
    94/100 stars

    Images

    1) 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

    Fbs1 GYR and PPRYR variants display reduced binding bias between high-mannose and complex N-glycans. ( a ) Comparison of N-glycoprotein pulldown by wt Fbs1, Fbs1 GYR and PPRYR variant proteins. A mixture of denatured RNase B and fetuin was subjected to an Fbs1 pulldown assay with wt, GYR and PPRYR Fbs1 beads in low salt (50 mM ammonium acetate, pH7.5) and high salt (2M ammonium acetate, pH7.5). All three Fbs1 bead types were conjugated with the same amount of the respective Fbs1 protein ( Supplementary Fig. 1 ). Left panel is the SDS–PAGE gel showing the bound (Lanes 1–6) and input ratio (Lane 7) of RNase B and fetuin. An asterisk denotes the SNAP-Fbs1 protein leaching from the Fbs1 beads. Right panel shows the recovery percentage (bound protein amount/input protein amount) of each substrate glycoprotein using the different conditions. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 GYR variant binding to a diverse set of N-glycopeptides is substantially unbiased. The experiment in Fig. 1d was repeated using Fbs1 GYR beads. The data shown in Fig. 1d are presented in this figure to facilitate the comparison between wt Fbs1 and Fbs1 GYR. N-glycans of SGP-TMR (1) were trimmed with different combinations of exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Identities of the trimmed SGP-TMR derivatives were confirmed by LC-MS. The trimmed glycopeptides were then added to binding assays with wt Fbs1 or Fbs1 GYR beads in 50 mM ammonium acetate pH7.5. The relative binding affinity to wt Fbs1 or Fbs1 GYR is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is not shown in the N-glycopeptide structures (1–4). Results represent the mean±s.e.m. of three replicates.
    Figure Legend Snippet: Fbs1 GYR and PPRYR variants display reduced binding bias between high-mannose and complex N-glycans. ( a ) Comparison of N-glycoprotein pulldown by wt Fbs1, Fbs1 GYR and PPRYR variant proteins. A mixture of denatured RNase B and fetuin was subjected to an Fbs1 pulldown assay with wt, GYR and PPRYR Fbs1 beads in low salt (50 mM ammonium acetate, pH7.5) and high salt (2M ammonium acetate, pH7.5). All three Fbs1 bead types were conjugated with the same amount of the respective Fbs1 protein ( Supplementary Fig. 1 ). Left panel is the SDS–PAGE gel showing the bound (Lanes 1–6) and input ratio (Lane 7) of RNase B and fetuin. An asterisk denotes the SNAP-Fbs1 protein leaching from the Fbs1 beads. Right panel shows the recovery percentage (bound protein amount/input protein amount) of each substrate glycoprotein using the different conditions. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 GYR variant binding to a diverse set of N-glycopeptides is substantially unbiased. The experiment in Fig. 1d was repeated using Fbs1 GYR beads. The data shown in Fig. 1d are presented in this figure to facilitate the comparison between wt Fbs1 and Fbs1 GYR. N-glycans of SGP-TMR (1) were trimmed with different combinations of exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Identities of the trimmed SGP-TMR derivatives were confirmed by LC-MS. The trimmed glycopeptides were then added to binding assays with wt Fbs1 or Fbs1 GYR beads in 50 mM ammonium acetate pH7.5. The relative binding affinity to wt Fbs1 or Fbs1 GYR is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is not shown in the N-glycopeptide structures (1–4). Results represent the mean±s.e.m. of three replicates.

    Techniques Used: Binding Assay, Variant Assay, SDS Page, Liquid Chromatography with Mass Spectroscopy, Fluorescence

    2) Product Images from "High-throughput analysis of N-glycans using AutoTip via glycoprotein immobilization"

    Article Title: High-throughput analysis of N-glycans using AutoTip via glycoprotein immobilization

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-10487-8

    Modification of sialic acids using carbodiimide coupling on AutoTip. Fetuin protein was modified by p-Toluidine in the presence of EDC after immobilization on the beads (Four AutoTips). Sialylated N-glycan, S2H5N4, was used to monitor completion of p-Toluidine-EDC reaction. Without derivatization, MALDI-MS detected no sialic acid H5N4 ( a ) and two sialic acids S2H5N4 ( c ); For incomplete derivatization, MALDI-MS detected ( a ), ( c ), and p-Toluidine labeled two sialic acids S2H5N4 ( b ); When completely labeled, only ( b ) was detected by MALDI-MS.
    Figure Legend Snippet: Modification of sialic acids using carbodiimide coupling on AutoTip. Fetuin protein was modified by p-Toluidine in the presence of EDC after immobilization on the beads (Four AutoTips). Sialylated N-glycan, S2H5N4, was used to monitor completion of p-Toluidine-EDC reaction. Without derivatization, MALDI-MS detected no sialic acid H5N4 ( a ) and two sialic acids S2H5N4 ( c ); For incomplete derivatization, MALDI-MS detected ( a ), ( c ), and p-Toluidine labeled two sialic acids S2H5N4 ( b ); When completely labeled, only ( b ) was detected by MALDI-MS.

    Techniques Used: Modification, Mass Spectrometry, Labeling

    3) 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

    Fbs1 GYR and PPRYR variants display reduced binding bias between high-mannose and complex N-glycans. ( a ) Comparison of N-glycoprotein pulldown by wt Fbs1, Fbs1 GYR and PPRYR variant proteins. A mixture of denatured RNase B and fetuin was subjected to an Fbs1 pulldown assay with wt, GYR and PPRYR Fbs1 beads in low salt (50 mM ammonium acetate, pH7.5) and high salt (2M ammonium acetate, pH7.5). All three Fbs1 bead types were conjugated with the same amount of the respective Fbs1 protein ( Supplementary Fig. 1 ). Left panel is the SDS–PAGE gel showing the bound (Lanes 1–6) and input ratio (Lane 7) of RNase B and fetuin. An asterisk denotes the SNAP-Fbs1 protein leaching from the Fbs1 beads. Right panel shows the recovery percentage (bound protein amount/input protein amount) of each substrate glycoprotein using the different conditions. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 GYR variant binding to a diverse set of N-glycopeptides is substantially unbiased. The experiment in Fig. 1d was repeated using Fbs1 GYR beads. The data shown in Fig. 1d are presented in this figure to facilitate the comparison between wt Fbs1 and Fbs1 GYR. N-glycans of SGP-TMR (1) were trimmed with different combinations of exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Identities of the trimmed SGP-TMR derivatives were confirmed by LC-MS. The trimmed glycopeptides were then added to binding assays with wt Fbs1 or Fbs1 GYR beads in 50 mM ammonium acetate pH7.5. The relative binding affinity to wt Fbs1 or Fbs1 GYR is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is not shown in the N-glycopeptide structures (1–4). Results represent the mean±s.e.m. of three replicates.
    Figure Legend Snippet: Fbs1 GYR and PPRYR variants display reduced binding bias between high-mannose and complex N-glycans. ( a ) Comparison of N-glycoprotein pulldown by wt Fbs1, Fbs1 GYR and PPRYR variant proteins. A mixture of denatured RNase B and fetuin was subjected to an Fbs1 pulldown assay with wt, GYR and PPRYR Fbs1 beads in low salt (50 mM ammonium acetate, pH7.5) and high salt (2M ammonium acetate, pH7.5). All three Fbs1 bead types were conjugated with the same amount of the respective Fbs1 protein ( Supplementary Fig. 1 ). Left panel is the SDS–PAGE gel showing the bound (Lanes 1–6) and input ratio (Lane 7) of RNase B and fetuin. An asterisk denotes the SNAP-Fbs1 protein leaching from the Fbs1 beads. Right panel shows the recovery percentage (bound protein amount/input protein amount) of each substrate glycoprotein using the different conditions. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 GYR variant binding to a diverse set of N-glycopeptides is substantially unbiased. The experiment in Fig. 1d was repeated using Fbs1 GYR beads. The data shown in Fig. 1d are presented in this figure to facilitate the comparison between wt Fbs1 and Fbs1 GYR. N-glycans of SGP-TMR (1) were trimmed with different combinations of exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Identities of the trimmed SGP-TMR derivatives were confirmed by LC-MS. The trimmed glycopeptides were then added to binding assays with wt Fbs1 or Fbs1 GYR beads in 50 mM ammonium acetate pH7.5. The relative binding affinity to wt Fbs1 or Fbs1 GYR is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is not shown in the N-glycopeptide structures (1–4). Results represent the mean±s.e.m. of three replicates.

    Techniques Used: Binding Assay, Variant Assay, SDS Page, Liquid Chromatography with Mass Spectroscopy, Fluorescence

    4) Product Images from "Brain clusterin protein isoforms and mitochondrial localization"

    Article Title: Brain clusterin protein isoforms and mitochondrial localization

    Journal: eLife

    doi: 10.7554/eLife.48255

    Positive controls for deglycosylation studies. RNase B, a high mannose glycoprotein, has a single N-linked glycosylation site and was used as a positive control for endoglycosidases that cleave N-linked carbohydrates. Fetuin, a glycoprotein containing sialylated N-linked and O-linked glycans, was used as a positive control for endoglycosidases that cleave both N-linked and O-linked carbohydrates.
    Figure Legend Snippet: Positive controls for deglycosylation studies. RNase B, a high mannose glycoprotein, has a single N-linked glycosylation site and was used as a positive control for endoglycosidases that cleave N-linked carbohydrates. Fetuin, a glycoprotein containing sialylated N-linked and O-linked glycans, was used as a positive control for endoglycosidases that cleave both N-linked and O-linked carbohydrates.

    Techniques Used: Positive Control

    5) Product Images from "Letter to the Glyco-Forum: Effective glycoanalysis with Maackia amurensis lectins requires a clear understanding of their binding specificities"

    Article Title: Letter to the Glyco-Forum: Effective glycoanalysis with Maackia amurensis lectins requires a clear understanding of their binding specificities

    Journal: Glycobiology

    doi: 10.1093/glycob/cwr080

    Lectin blots of untreated (−), S. typhimurium α2-3 sialidase treated (α2-3 N) or PNGase F (PNG-F) treated bovine fetuin. CBB, Coomassie brilliant blue stain; MAL, Maackia amurensis leukoagglutinin; MAH, Maackia amurensis hemagglutinin;
    Figure Legend Snippet: Lectin blots of untreated (−), S. typhimurium α2-3 sialidase treated (α2-3 N) or PNGase F (PNG-F) treated bovine fetuin. CBB, Coomassie brilliant blue stain; MAL, Maackia amurensis leukoagglutinin; MAH, Maackia amurensis hemagglutinin;

    Techniques Used: Staining

    6) Product Images from "Brain clusterin protein isoforms and mitochondrial localization"

    Article Title: Brain clusterin protein isoforms and mitochondrial localization

    Journal: eLife

    doi: 10.7554/eLife.48255

    Positive controls for deglycosylation studies. RNase B, a high mannose glycoprotein, has a single N-linked glycosylation site and was used as a positive control for endoglycosidases that cleave N-linked carbohydrates. Fetuin, a glycoprotein containing sialylated N-linked and O-linked glycans, was used as a positive control for endoglycosidases that cleave both N-linked and O-linked carbohydrates.
    Figure Legend Snippet: Positive controls for deglycosylation studies. RNase B, a high mannose glycoprotein, has a single N-linked glycosylation site and was used as a positive control for endoglycosidases that cleave N-linked carbohydrates. Fetuin, a glycoprotein containing sialylated N-linked and O-linked glycans, was used as a positive control for endoglycosidases that cleave both N-linked and O-linked carbohydrates.

    Techniques Used: Positive Control

    7) Product Images from "Letter to the Glyco-Forum: Effective glycoanalysis with Maackia amurensis lectins requires a clear understanding of their binding specificities"

    Article Title: Letter to the Glyco-Forum: Effective glycoanalysis with Maackia amurensis lectins requires a clear understanding of their binding specificities

    Journal: Glycobiology

    doi: 10.1093/glycob/cwr080

    Lectin blots of untreated (−), S. typhimurium α2-3 sialidase treated (α2-3 N) or PNGase F (PNG-F) treated bovine fetuin. CBB, Coomassie brilliant blue stain; MAL, Maackia amurensis leukoagglutinin; MAH, Maackia amurensis hemagglutinin;
    Figure Legend Snippet: Lectin blots of untreated (−), S. typhimurium α2-3 sialidase treated (α2-3 N) or PNGase F (PNG-F) treated bovine fetuin. CBB, Coomassie brilliant blue stain; MAL, Maackia amurensis leukoagglutinin; MAH, Maackia amurensis hemagglutinin;

    Techniques Used: Staining

    Related Articles

    High Performance Liquid Chromatography:

    Article Title: High-throughput analysis of N-glycans using AutoTip via glycoprotein immobilization
    Article Snippet: .. Each AutoTip conjugated 20 µg of fetuin after denaturation (88 µL fetuin protein in HPLC water +10 µL denaturing buffer (NEB); 100 °C/10 min). .. N-glycans were released after sialic acid modification by PNGase F at room temperature (2 h), and subjected to desalting using Hypercarb AutoTips.

    Activity Assay:

    Article Title: Biochemical and functional characterization of CpMuc4, aCryptosporidium surface antigen that binds to host epithelial cells
    Article Snippet: .. The activity of the enzymes in the ProteinDeglycosylation Mix was confirmed by digestion of fetuin, an N- andO-glycosylated protein included in the protein deglycosylation kit as a positivecontrol (NEB). .. The effect of the glycosidases on the apparentmolecular weight of nCpMuc4 was evaluated by western blot.

    Article Title: Overexpression of GLT1D1 induces immunosuppression through glycosylation of PD‐L1 and predicts poor prognosis in B‐cell lymphoma
    Article Snippet: .. We also used fetuin, a glycoprotein containing sialylated N‐linked and O‐linked glycans, as a positive control for the PNGase F enzyme activity, using an assay kit from New England BioLabs (Ipswich, MA, USA). .. Incubation of fetuin with PNGase F caused an almost complete cleavage of the N‐linked oligosaccharides, thus shifting the fetuin band to a lower molecular weight position (Fig. A).

    Positive Control:

    Article Title: Overexpression of GLT1D1 induces immunosuppression through glycosylation of PD‐L1 and predicts poor prognosis in B‐cell lymphoma
    Article Snippet: .. We also used fetuin, a glycoprotein containing sialylated N‐linked and O‐linked glycans, as a positive control for the PNGase F enzyme activity, using an assay kit from New England BioLabs (Ipswich, MA, USA). .. Incubation of fetuin with PNGase F caused an almost complete cleavage of the N‐linked oligosaccharides, thus shifting the fetuin band to a lower molecular weight position (Fig. A).

    Mass Spectrometry:

    Article Title: An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides
    Article Snippet: .. Materials RNase B, fetuin, PNGase F, α2-3,6,8 Neuraminidase, β1-4 Galactosidase S, β-N-Acetylglucosaminidase S and MS-grade Trypsin were from New England Biolabs (NEB) (Ipswich, MA). .. Human IgG was purchased from Bethyl Laboratories, Inc. (Montgomery, TX).

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    New England Biolabs fetuin
    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 <t>fetuin</t> relative to <t>RNase</t> 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.
    Fetuin, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.

    Journal: Nature Communications

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

    doi: 10.1038/ncomms15487

    Figure Lengend 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.

    Article Snippet: Materials RNase B, fetuin, PNGase F, α2-3,6,8 Neuraminidase, β1-4 Galactosidase S, β-N-Acetylglucosaminidase S and MS-grade Trypsin were from New England Biolabs (NEB) (Ipswich, MA).

    Techniques: Binding Assay, Sequencing, Incubation, SDS Page

    Fbs1 GYR and PPRYR variants display reduced binding bias between high-mannose and complex N-glycans. ( a ) Comparison of N-glycoprotein pulldown by wt Fbs1, Fbs1 GYR and PPRYR variant proteins. A mixture of denatured RNase B and fetuin was subjected to an Fbs1 pulldown assay with wt, GYR and PPRYR Fbs1 beads in low salt (50 mM ammonium acetate, pH7.5) and high salt (2M ammonium acetate, pH7.5). All three Fbs1 bead types were conjugated with the same amount of the respective Fbs1 protein ( Supplementary Fig. 1 ). Left panel is the SDS–PAGE gel showing the bound (Lanes 1–6) and input ratio (Lane 7) of RNase B and fetuin. An asterisk denotes the SNAP-Fbs1 protein leaching from the Fbs1 beads. Right panel shows the recovery percentage (bound protein amount/input protein amount) of each substrate glycoprotein using the different conditions. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 GYR variant binding to a diverse set of N-glycopeptides is substantially unbiased. The experiment in Fig. 1d was repeated using Fbs1 GYR beads. The data shown in Fig. 1d are presented in this figure to facilitate the comparison between wt Fbs1 and Fbs1 GYR. N-glycans of SGP-TMR (1) were trimmed with different combinations of exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Identities of the trimmed SGP-TMR derivatives were confirmed by LC-MS. The trimmed glycopeptides were then added to binding assays with wt Fbs1 or Fbs1 GYR beads in 50 mM ammonium acetate pH7.5. The relative binding affinity to wt Fbs1 or Fbs1 GYR is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is not shown in the N-glycopeptide structures (1–4). Results represent the mean±s.e.m. of three replicates.

    Journal: Nature Communications

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

    doi: 10.1038/ncomms15487

    Figure Lengend Snippet: Fbs1 GYR and PPRYR variants display reduced binding bias between high-mannose and complex N-glycans. ( a ) Comparison of N-glycoprotein pulldown by wt Fbs1, Fbs1 GYR and PPRYR variant proteins. A mixture of denatured RNase B and fetuin was subjected to an Fbs1 pulldown assay with wt, GYR and PPRYR Fbs1 beads in low salt (50 mM ammonium acetate, pH7.5) and high salt (2M ammonium acetate, pH7.5). All three Fbs1 bead types were conjugated with the same amount of the respective Fbs1 protein ( Supplementary Fig. 1 ). Left panel is the SDS–PAGE gel showing the bound (Lanes 1–6) and input ratio (Lane 7) of RNase B and fetuin. An asterisk denotes the SNAP-Fbs1 protein leaching from the Fbs1 beads. Right panel shows the recovery percentage (bound protein amount/input protein amount) of each substrate glycoprotein using the different conditions. A representative SDS–PAGE gel is shown from two experiments. ( b ) Fbs1 GYR variant binding to a diverse set of N-glycopeptides is substantially unbiased. The experiment in Fig. 1d was repeated using Fbs1 GYR beads. The data shown in Fig. 1d are presented in this figure to facilitate the comparison between wt Fbs1 and Fbs1 GYR. N-glycans of SGP-TMR (1) were trimmed with different combinations of exoglycosidases to produce asialo-SGP-TMR (2), SGP-TMR without sialic acids and galactose (3) and SGP-TMR without sialic acids, galactose and GlcNAc (4). Identities of the trimmed SGP-TMR derivatives were confirmed by LC-MS. The trimmed glycopeptides were then added to binding assays with wt Fbs1 or Fbs1 GYR beads in 50 mM ammonium acetate pH7.5. The relative binding affinity to wt Fbs1 or Fbs1 GYR is reported as the recovery percentage (TMR fluorescence on beads/input TMR fluorescence). For simplicity, TMR is not shown in the N-glycopeptide structures (1–4). Results represent the mean±s.e.m. of three replicates.

    Article Snippet: Materials RNase B, fetuin, PNGase F, α2-3,6,8 Neuraminidase, β1-4 Galactosidase S, β-N-Acetylglucosaminidase S and MS-grade Trypsin were from New England Biolabs (NEB) (Ipswich, MA).

    Techniques: Binding Assay, Variant Assay, SDS Page, Liquid Chromatography with Mass Spectroscopy, Fluorescence

    Modification of sialic acids using carbodiimide coupling on AutoTip. Fetuin protein was modified by p-Toluidine in the presence of EDC after immobilization on the beads (Four AutoTips). Sialylated N-glycan, S2H5N4, was used to monitor completion of p-Toluidine-EDC reaction. Without derivatization, MALDI-MS detected no sialic acid H5N4 ( a ) and two sialic acids S2H5N4 ( c ); For incomplete derivatization, MALDI-MS detected ( a ), ( c ), and p-Toluidine labeled two sialic acids S2H5N4 ( b ); When completely labeled, only ( b ) was detected by MALDI-MS.

    Journal: Scientific Reports

    Article Title: High-throughput analysis of N-glycans using AutoTip via glycoprotein immobilization

    doi: 10.1038/s41598-017-10487-8

    Figure Lengend Snippet: Modification of sialic acids using carbodiimide coupling on AutoTip. Fetuin protein was modified by p-Toluidine in the presence of EDC after immobilization on the beads (Four AutoTips). Sialylated N-glycan, S2H5N4, was used to monitor completion of p-Toluidine-EDC reaction. Without derivatization, MALDI-MS detected no sialic acid H5N4 ( a ) and two sialic acids S2H5N4 ( c ); For incomplete derivatization, MALDI-MS detected ( a ), ( c ), and p-Toluidine labeled two sialic acids S2H5N4 ( b ); When completely labeled, only ( b ) was detected by MALDI-MS.

    Article Snippet: Each AutoTip conjugated 20 µg of fetuin after denaturation (88 µL fetuin protein in HPLC water +10 µL denaturing buffer (NEB); 100 °C/10 min).

    Techniques: Modification, Mass Spectrometry, Labeling

    Lectin blots of untreated (−), S. typhimurium α2-3 sialidase treated (α2-3 N) or PNGase F (PNG-F) treated bovine fetuin. CBB, Coomassie brilliant blue stain; MAL, Maackia amurensis leukoagglutinin; MAH, Maackia amurensis hemagglutinin;

    Journal: Glycobiology

    Article Title: Letter to the Glyco-Forum: Effective glycoanalysis with Maackia amurensis lectins requires a clear understanding of their binding specificities

    doi: 10.1093/glycob/cwr080

    Figure Lengend Snippet: Lectin blots of untreated (−), S. typhimurium α2-3 sialidase treated (α2-3 N) or PNGase F (PNG-F) treated bovine fetuin. CBB, Coomassie brilliant blue stain; MAL, Maackia amurensis leukoagglutinin; MAH, Maackia amurensis hemagglutinin;

    Article Snippet: For desialylation, samples of the fetuin solution were treated with Salmonella typhimurium α2-3 sialidase (New England Biolabs, Ipswich, MA) according to the manufacturer's instructions.

    Techniques: Staining