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    Name:
    Remove iT PNGase F
    Description:
    Remove iT PNGase F 33 750 units
    Catalog Number:
    p0706l
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    33 750 units
    Category:
    Glycosidases
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    Structured Review

    New England Biolabs cbd pngase f
    Remove iT PNGase F
    Remove iT PNGase F 33 750 units
    https://www.bioz.com/result/cbd pngase f/product/New England Biolabs
    Average 97 stars, based on 43 article reviews
    Price from $9.99 to $1999.99
    cbd pngase f - by Bioz Stars, 2020-08
    97/100 stars

    Images

    1) Product Images from "Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens"

    Article Title: Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens

    Journal: Journal of proteome research

    doi: 10.1021/acs.jproteome.9b00620

    N-Glycanation as initially identified as spontaneous reformation of amidase substrate following cleavage by PNGase F, a reaction that could be cycled indefinitely. (A) HA was de-N-glycosylated using chitin binding domain (CBD)-PNGase F, which when removed by magnetic chitin protein beads was concomitant with recovery in HA’s electrophoretic mobility (GelCode Blue staining), glycan stain (PAS) (pink), and reacquired sensitivity to CBD-PNGase F (three iterative cycles of de-N-glycosylation by amidase exposure + recovery after amidase removal are shown). (B) HA and ‘glycan recovered HA’ were also further separated by SEC (Superdex 200 10/300 column). (C) The recombinant HA was co-expressed with NA to cleave terminal SA, to prevent aggregation in solution. To assess the reformation of amidase substrate when terminal SA was present, Y98F HA trimer was used (GelCode Blue stain and western blot using SNA to detect SA).
    Figure Legend Snippet: N-Glycanation as initially identified as spontaneous reformation of amidase substrate following cleavage by PNGase F, a reaction that could be cycled indefinitely. (A) HA was de-N-glycosylated using chitin binding domain (CBD)-PNGase F, which when removed by magnetic chitin protein beads was concomitant with recovery in HA’s electrophoretic mobility (GelCode Blue staining), glycan stain (PAS) (pink), and reacquired sensitivity to CBD-PNGase F (three iterative cycles of de-N-glycosylation by amidase exposure + recovery after amidase removal are shown). (B) HA and ‘glycan recovered HA’ were also further separated by SEC (Superdex 200 10/300 column). (C) The recombinant HA was co-expressed with NA to cleave terminal SA, to prevent aggregation in solution. To assess the reformation of amidase substrate when terminal SA was present, Y98F HA trimer was used (GelCode Blue stain and western blot using SNA to detect SA).

    Techniques Used: Binding Assay, Staining, Recombinant, Western Blot

    2) Product Images from "Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens"

    Article Title: Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens

    Journal: Journal of proteome research

    doi: 10.1021/acs.jproteome.9b00620

    N-Glycanation as initially identified as spontaneous reformation of amidase substrate following cleavage by PNGase F, a reaction that could be cycled indefinitely. (A) HA was de-N-glycosylated using chitin binding domain (CBD)-PNGase F, which when removed by magnetic chitin protein beads was concomitant with recovery in HA’s electrophoretic mobility (GelCode Blue staining), glycan stain (PAS) (pink), and reacquired sensitivity to CBD-PNGase F (three iterative cycles of de-N-glycosylation by amidase exposure + recovery after amidase removal are shown). (B) HA and ‘glycan recovered HA’ were also further separated by SEC (Superdex 200 10/300 column). (C) The recombinant HA was co-expressed with NA to cleave terminal SA, to prevent aggregation in solution. To assess the reformation of amidase substrate when terminal SA was present, Y98F HA trimer was used (GelCode Blue stain and western blot using SNA to detect SA).
    Figure Legend Snippet: N-Glycanation as initially identified as spontaneous reformation of amidase substrate following cleavage by PNGase F, a reaction that could be cycled indefinitely. (A) HA was de-N-glycosylated using chitin binding domain (CBD)-PNGase F, which when removed by magnetic chitin protein beads was concomitant with recovery in HA’s electrophoretic mobility (GelCode Blue staining), glycan stain (PAS) (pink), and reacquired sensitivity to CBD-PNGase F (three iterative cycles of de-N-glycosylation by amidase exposure + recovery after amidase removal are shown). (B) HA and ‘glycan recovered HA’ were also further separated by SEC (Superdex 200 10/300 column). (C) The recombinant HA was co-expressed with NA to cleave terminal SA, to prevent aggregation in solution. To assess the reformation of amidase substrate when terminal SA was present, Y98F HA trimer was used (GelCode Blue stain and western blot using SNA to detect SA).

    Techniques Used: Binding Assay, Staining, Recombinant, Western Blot

    N-glycanation is preceded by non-covalent retention of cleaved N-glycan. (A) HA was de-N-glycosylated by PNGase F and the mixture was separated by SDS PAGE or native PAGE without prior removal of PNGase F. The gels were stained with PAS to mark glycan (pink) and also GelCode Blue for protein. (B) To further define non-covalent retention of N-glycan following cleavage by PNGase F, the HA+ PNGase F de-N-glycosylation reaction was immediately separated by SEC (Superdex 200 10/300 column; in this case no chitin beads affinity beads to pre-remove the amidase from solution). (C) HA product of this SEC was then analyzed for N-glycanation by SDS-PAGE (GelCode blue for protein, PAS to mark glycan and re-exposure to PNGase F to mark reformation of amidase substrate).
    Figure Legend Snippet: N-glycanation is preceded by non-covalent retention of cleaved N-glycan. (A) HA was de-N-glycosylated by PNGase F and the mixture was separated by SDS PAGE or native PAGE without prior removal of PNGase F. The gels were stained with PAS to mark glycan (pink) and also GelCode Blue for protein. (B) To further define non-covalent retention of N-glycan following cleavage by PNGase F, the HA+ PNGase F de-N-glycosylation reaction was immediately separated by SEC (Superdex 200 10/300 column; in this case no chitin beads affinity beads to pre-remove the amidase from solution). (C) HA product of this SEC was then analyzed for N-glycanation by SDS-PAGE (GelCode blue for protein, PAS to mark glycan and re-exposure to PNGase F to mark reformation of amidase substrate).

    Techniques Used: SDS Page, Clear Native PAGE, Staining

    A non-enzymatic but conformational requirement for glycan retention and N-glycanation. (A) If HA was denatured (100°C for 30 min), it no longer retained glycan in the presence of PNGase F, as marked by native PAGE followed by GelCode Blue stain for protein (blue) and PAS stain for glycan (pink). (B) Heat denatured HA also no longer served as substrate for N-glycanation, as evaluated by de-N-glycosylation by PNGase F followed by SEC and SDS PAGE and GelCode Blue staining. Lanes 1–3 are for a control N-glycanation reaction (1= HA; 2= HA + PNGase F; 3 = HA recovered post PNGase F treatment and post separation by SEC). Lanes 4–6 denote the same reaction except with heat denatured HA as substrate (4= HA; 5 = HA + PNGase F; 6 = HA recovered post PNGase F treatment and post separation by SEC). (C) Denatured HA was mixed with folded trimeric HA and the mixture was de-N-glycosylated by PNGase F and the capacity for N-glycanation was evaluated by SDS PAGE (GelCode Blue stain) before and after separation by SEC (Superdex 200 10/300 column). Both HA and denatured HA could be separated by SEC. Both HA forms also underwent de-N-glycosylation together (shift in MW in the SDS PAGE readout), but only denatured HA fraction remained de-N-glycosylated after chromatographic separation from the amidase. HA recovered from the trimeric elution volume on SEC had now required the glycan. (D) Upper gel: we confirm Mn 2+ as an inhibitor of deglycosylation activity by PNGase F. The deglycosylation reaction was performed at 37°C for 24 h in the presence/absence of Mn 2+ (all in absence of metal ion chelators) and visualized by SDS PAGE with GelCode Blue stain). Lower gel: N-glycanation in the presence of Mn 2+ . HA was deglycosylated by PNGase F in the absence of metal ion chelators (50nM Tris, pH 8.0) and then adjusted (treated) or not adjusted (control) to 1 mM MnCl 2 . The control and treated reactions were then separated by SEC where the column and FPLC system was pre-equilibrated in 50 mM Tris, pH 8.0 ± MnCl 2 . In both cases a single peak corresponding to the HA trimer was collected and then visualized by SDS PAGE with GelCode Blue stain.
    Figure Legend Snippet: A non-enzymatic but conformational requirement for glycan retention and N-glycanation. (A) If HA was denatured (100°C for 30 min), it no longer retained glycan in the presence of PNGase F, as marked by native PAGE followed by GelCode Blue stain for protein (blue) and PAS stain for glycan (pink). (B) Heat denatured HA also no longer served as substrate for N-glycanation, as evaluated by de-N-glycosylation by PNGase F followed by SEC and SDS PAGE and GelCode Blue staining. Lanes 1–3 are for a control N-glycanation reaction (1= HA; 2= HA + PNGase F; 3 = HA recovered post PNGase F treatment and post separation by SEC). Lanes 4–6 denote the same reaction except with heat denatured HA as substrate (4= HA; 5 = HA + PNGase F; 6 = HA recovered post PNGase F treatment and post separation by SEC). (C) Denatured HA was mixed with folded trimeric HA and the mixture was de-N-glycosylated by PNGase F and the capacity for N-glycanation was evaluated by SDS PAGE (GelCode Blue stain) before and after separation by SEC (Superdex 200 10/300 column). Both HA and denatured HA could be separated by SEC. Both HA forms also underwent de-N-glycosylation together (shift in MW in the SDS PAGE readout), but only denatured HA fraction remained de-N-glycosylated after chromatographic separation from the amidase. HA recovered from the trimeric elution volume on SEC had now required the glycan. (D) Upper gel: we confirm Mn 2+ as an inhibitor of deglycosylation activity by PNGase F. The deglycosylation reaction was performed at 37°C for 24 h in the presence/absence of Mn 2+ (all in absence of metal ion chelators) and visualized by SDS PAGE with GelCode Blue stain). Lower gel: N-glycanation in the presence of Mn 2+ . HA was deglycosylated by PNGase F in the absence of metal ion chelators (50nM Tris, pH 8.0) and then adjusted (treated) or not adjusted (control) to 1 mM MnCl 2 . The control and treated reactions were then separated by SEC where the column and FPLC system was pre-equilibrated in 50 mM Tris, pH 8.0 ± MnCl 2 . In both cases a single peak corresponding to the HA trimer was collected and then visualized by SDS PAGE with GelCode Blue stain.

    Techniques Used: Clear Native PAGE, Staining, SDS Page, Activity Assay, Fast Protein Liquid Chromatography

    3) Product Images from "Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens"

    Article Title: Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens

    Journal: Journal of proteome research

    doi: 10.1021/acs.jproteome.9b00620

    N-Glycanation as initially identified as spontaneous reformation of amidase substrate following cleavage by PNGase F, a reaction that could be cycled indefinitely. (A) HA was de-N-glycosylated using chitin binding domain (CBD)-PNGase F, which when removed by magnetic chitin protein beads was concomitant with recovery in HA’s electrophoretic mobility (GelCode Blue staining), glycan stain (PAS) (pink), and reacquired sensitivity to CBD-PNGase F (three iterative cycles of de-N-glycosylation by amidase exposure + recovery after amidase removal are shown). (B) HA and ‘glycan recovered HA’ were also further separated by SEC (Superdex 200 10/300 column). (C) The recombinant HA was co-expressed with NA to cleave terminal SA, to prevent aggregation in solution. To assess the reformation of amidase substrate when terminal SA was present, Y98F HA trimer was used (GelCode Blue stain and western blot using SNA to detect SA).
    Figure Legend Snippet: N-Glycanation as initially identified as spontaneous reformation of amidase substrate following cleavage by PNGase F, a reaction that could be cycled indefinitely. (A) HA was de-N-glycosylated using chitin binding domain (CBD)-PNGase F, which when removed by magnetic chitin protein beads was concomitant with recovery in HA’s electrophoretic mobility (GelCode Blue staining), glycan stain (PAS) (pink), and reacquired sensitivity to CBD-PNGase F (three iterative cycles of de-N-glycosylation by amidase exposure + recovery after amidase removal are shown). (B) HA and ‘glycan recovered HA’ were also further separated by SEC (Superdex 200 10/300 column). (C) The recombinant HA was co-expressed with NA to cleave terminal SA, to prevent aggregation in solution. To assess the reformation of amidase substrate when terminal SA was present, Y98F HA trimer was used (GelCode Blue stain and western blot using SNA to detect SA).

    Techniques Used: Binding Assay, Staining, Recombinant, Western Blot

    4) Product Images from "Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens"

    Article Title: Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens

    Journal: Journal of proteome research

    doi: 10.1021/acs.jproteome.9b00620

    N-Glycanation as initially identified as spontaneous reformation of amidase substrate following cleavage by PNGase F, a reaction that could be cycled indefinitely. (A) HA was de-N-glycosylated using chitin binding domain (CBD)-PNGase F, which when removed by magnetic chitin protein beads was concomitant with recovery in HA’s electrophoretic mobility (GelCode Blue staining), glycan stain (PAS) (pink), and reacquired sensitivity to CBD-PNGase F (three iterative cycles of de-N-glycosylation by amidase exposure + recovery after amidase removal are shown). (B) HA and ‘glycan recovered HA’ were also further separated by SEC (Superdex 200 10/300 column). (C) The recombinant HA was co-expressed with NA to cleave terminal SA, to prevent aggregation in solution. To assess the reformation of amidase substrate when terminal SA was present, Y98F HA trimer was used (GelCode Blue stain and western blot using SNA to detect SA).
    Figure Legend Snippet: N-Glycanation as initially identified as spontaneous reformation of amidase substrate following cleavage by PNGase F, a reaction that could be cycled indefinitely. (A) HA was de-N-glycosylated using chitin binding domain (CBD)-PNGase F, which when removed by magnetic chitin protein beads was concomitant with recovery in HA’s electrophoretic mobility (GelCode Blue staining), glycan stain (PAS) (pink), and reacquired sensitivity to CBD-PNGase F (three iterative cycles of de-N-glycosylation by amidase exposure + recovery after amidase removal are shown). (B) HA and ‘glycan recovered HA’ were also further separated by SEC (Superdex 200 10/300 column). (C) The recombinant HA was co-expressed with NA to cleave terminal SA, to prevent aggregation in solution. To assess the reformation of amidase substrate when terminal SA was present, Y98F HA trimer was used (GelCode Blue stain and western blot using SNA to detect SA).

    Techniques Used: Binding Assay, Staining, Recombinant, Western Blot

    N-glycanation is preceded by non-covalent retention of cleaved N-glycan. (A) HA was de-N-glycosylated by PNGase F and the mixture was separated by SDS PAGE or native PAGE without prior removal of PNGase F. The gels were stained with PAS to mark glycan (pink) and also GelCode Blue for protein. (B) To further define non-covalent retention of N-glycan following cleavage by PNGase F, the HA+ PNGase F de-N-glycosylation reaction was immediately separated by SEC (Superdex 200 10/300 column; in this case no chitin beads affinity beads to pre-remove the amidase from solution). (C) HA product of this SEC was then analyzed for N-glycanation by SDS-PAGE (GelCode blue for protein, PAS to mark glycan and re-exposure to PNGase F to mark reformation of amidase substrate).
    Figure Legend Snippet: N-glycanation is preceded by non-covalent retention of cleaved N-glycan. (A) HA was de-N-glycosylated by PNGase F and the mixture was separated by SDS PAGE or native PAGE without prior removal of PNGase F. The gels were stained with PAS to mark glycan (pink) and also GelCode Blue for protein. (B) To further define non-covalent retention of N-glycan following cleavage by PNGase F, the HA+ PNGase F de-N-glycosylation reaction was immediately separated by SEC (Superdex 200 10/300 column; in this case no chitin beads affinity beads to pre-remove the amidase from solution). (C) HA product of this SEC was then analyzed for N-glycanation by SDS-PAGE (GelCode blue for protein, PAS to mark glycan and re-exposure to PNGase F to mark reformation of amidase substrate).

    Techniques Used: SDS Page, Clear Native PAGE, Staining

    A non-enzymatic but conformational requirement for glycan retention and N-glycanation. (A) If HA was denatured (100°C for 30 min), it no longer retained glycan in the presence of PNGase F, as marked by native PAGE followed by GelCode Blue stain for protein (blue) and PAS stain for glycan (pink). (B) Heat denatured HA also no longer served as substrate for N-glycanation, as evaluated by de-N-glycosylation by PNGase F followed by SEC and SDS PAGE and GelCode Blue staining. Lanes 1–3 are for a control N-glycanation reaction (1= HA; 2= HA + PNGase F; 3 = HA recovered post PNGase F treatment and post separation by SEC). Lanes 4–6 denote the same reaction except with heat denatured HA as substrate (4= HA; 5 = HA + PNGase F; 6 = HA recovered post PNGase F treatment and post separation by SEC). (C) Denatured HA was mixed with folded trimeric HA and the mixture was de-N-glycosylated by PNGase F and the capacity for N-glycanation was evaluated by SDS PAGE (GelCode Blue stain) before and after separation by SEC (Superdex 200 10/300 column). Both HA and denatured HA could be separated by SEC. Both HA forms also underwent de-N-glycosylation together (shift in MW in the SDS PAGE readout), but only denatured HA fraction remained de-N-glycosylated after chromatographic separation from the amidase. HA recovered from the trimeric elution volume on SEC had now required the glycan. (D) Upper gel: we confirm Mn 2+ as an inhibitor of deglycosylation activity by PNGase F. The deglycosylation reaction was performed at 37°C for 24 h in the presence/absence of Mn 2+ (all in absence of metal ion chelators) and visualized by SDS PAGE with GelCode Blue stain). Lower gel: N-glycanation in the presence of Mn 2+ . HA was deglycosylated by PNGase F in the absence of metal ion chelators (50nM Tris, pH 8.0) and then adjusted (treated) or not adjusted (control) to 1 mM MnCl 2 . The control and treated reactions were then separated by SEC where the column and FPLC system was pre-equilibrated in 50 mM Tris, pH 8.0 ± MnCl 2 . In both cases a single peak corresponding to the HA trimer was collected and then visualized by SDS PAGE with GelCode Blue stain.
    Figure Legend Snippet: A non-enzymatic but conformational requirement for glycan retention and N-glycanation. (A) If HA was denatured (100°C for 30 min), it no longer retained glycan in the presence of PNGase F, as marked by native PAGE followed by GelCode Blue stain for protein (blue) and PAS stain for glycan (pink). (B) Heat denatured HA also no longer served as substrate for N-glycanation, as evaluated by de-N-glycosylation by PNGase F followed by SEC and SDS PAGE and GelCode Blue staining. Lanes 1–3 are for a control N-glycanation reaction (1= HA; 2= HA + PNGase F; 3 = HA recovered post PNGase F treatment and post separation by SEC). Lanes 4–6 denote the same reaction except with heat denatured HA as substrate (4= HA; 5 = HA + PNGase F; 6 = HA recovered post PNGase F treatment and post separation by SEC). (C) Denatured HA was mixed with folded trimeric HA and the mixture was de-N-glycosylated by PNGase F and the capacity for N-glycanation was evaluated by SDS PAGE (GelCode Blue stain) before and after separation by SEC (Superdex 200 10/300 column). Both HA and denatured HA could be separated by SEC. Both HA forms also underwent de-N-glycosylation together (shift in MW in the SDS PAGE readout), but only denatured HA fraction remained de-N-glycosylated after chromatographic separation from the amidase. HA recovered from the trimeric elution volume on SEC had now required the glycan. (D) Upper gel: we confirm Mn 2+ as an inhibitor of deglycosylation activity by PNGase F. The deglycosylation reaction was performed at 37°C for 24 h in the presence/absence of Mn 2+ (all in absence of metal ion chelators) and visualized by SDS PAGE with GelCode Blue stain). Lower gel: N-glycanation in the presence of Mn 2+ . HA was deglycosylated by PNGase F in the absence of metal ion chelators (50nM Tris, pH 8.0) and then adjusted (treated) or not adjusted (control) to 1 mM MnCl 2 . The control and treated reactions were then separated by SEC where the column and FPLC system was pre-equilibrated in 50 mM Tris, pH 8.0 ± MnCl 2 . In both cases a single peak corresponding to the HA trimer was collected and then visualized by SDS PAGE with GelCode Blue stain.

    Techniques Used: Clear Native PAGE, Staining, SDS Page, Activity Assay, Fast Protein Liquid Chromatography

    5) Product Images from "Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens"

    Article Title: Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens

    Journal: Journal of proteome research

    doi: 10.1021/acs.jproteome.9b00620

    N-Glycanation as initially identified as spontaneous reformation of amidase substrate following cleavage by PNGase F, a reaction that could be cycled indefinitely. (A) HA was de-N-glycosylated using chitin binding domain (CBD)-PNGase F, which when removed by magnetic chitin protein beads was concomitant with recovery in HA’s electrophoretic mobility (GelCode Blue staining), glycan stain (PAS) (pink), and reacquired sensitivity to CBD-PNGase F (three iterative cycles of de-N-glycosylation by amidase exposure + recovery after amidase removal are shown). (B) HA and ‘glycan recovered HA’ were also further separated by SEC (Superdex 200 10/300 column). (C) The recombinant HA was co-expressed with NA to cleave terminal SA, to prevent aggregation in solution. To assess the reformation of amidase substrate when terminal SA was present, Y98F HA trimer was used (GelCode Blue stain and western blot using SNA to detect SA).
    Figure Legend Snippet: N-Glycanation as initially identified as spontaneous reformation of amidase substrate following cleavage by PNGase F, a reaction that could be cycled indefinitely. (A) HA was de-N-glycosylated using chitin binding domain (CBD)-PNGase F, which when removed by magnetic chitin protein beads was concomitant with recovery in HA’s electrophoretic mobility (GelCode Blue staining), glycan stain (PAS) (pink), and reacquired sensitivity to CBD-PNGase F (three iterative cycles of de-N-glycosylation by amidase exposure + recovery after amidase removal are shown). (B) HA and ‘glycan recovered HA’ were also further separated by SEC (Superdex 200 10/300 column). (C) The recombinant HA was co-expressed with NA to cleave terminal SA, to prevent aggregation in solution. To assess the reformation of amidase substrate when terminal SA was present, Y98F HA trimer was used (GelCode Blue stain and western blot using SNA to detect SA).

    Techniques Used: Binding Assay, Staining, Recombinant, Western Blot

    Related Articles

    Magnetic Beads:

    Article Title: Identification of an ADAM17 Cleavage Region in Human CD16 (FcγRIII) and the Engineering of a Non-Cleavable Version of the Receptor in NK Cells
    Article Snippet: .. Remove-iT PNGase F was then removed from the reaction using chitin magnetic beads (New England BioLabs). .. CD16 was subjected to SDS-PAGE and gel bands corresponding to soluble CD16 were detected by a Krypton Fluorescent Protein Stain (Thermo Fisher Scientific), verified by CD16 immunoblot analysis of adjacent lanes in the same gel, and were then excised and subjected to standard in-gel digestion with trypsin.

    Purification:

    Article Title: Spontaneous Glycan Reattachment Following N-Glycanase Treatment of Influenza and HIV Vaccine Antigens.
    Article Snippet: .. In cells, asparagine/N-linked glycans are added to glycoproteins cotranslationally, in an attachment process that supports proper folding of the nascent polypeptide. .. In cells, asparagine/N-linked glycans are added to glycoproteins cotranslationally, in an attachment process that supports proper folding of the nascent polypeptide.

    Article Title: Kinetic analysis and structural studies of a high‐efficiency laccase from Cerrena sp. RSD1
    Article Snippet: .. For deglycosylation assays , purified DLac was treated with either Endo H (New England BioLabs) or Remove‐iT™ PNGase F (New England BioLabs) for 24 h at 37 °C according to the manufacturer's instructions. .. The residual activity was measured by standard enzymatic assay.

    Article Title: Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens
    Article Snippet: .. Purified influenza H1 HA trimer was first de-N-glycosylated under non-denaturing conditions using commercial PNGase F tagged with a chitin-binding domain (CBD) (Remove-iT® PNGase F, Cat# P0706S, New England BioLabs) (=CBD-PNGase F) according to the manufacturer’s instructions (0.72 mg/mL HA, 40 000 units/mL CBD-PNGase F, 50 mM Na3 PO4 , pH 7.5; 24 h incubation at 37°C). .. Chitin magnetic beads (Cat# E8036S, New England BioLabs) equilibrated CBD binding buffer (500 mM NaCl, 20 mM Tris-HCI, 1 mM EDTA, pH 8) were incubated with the deglycosylation reaction (1 hr at 4°C, RotoFlex Plus Tube Rotator, Argos Technologies) and then applied to deplete CBD-PNGase from solution via separation on a magnetic column (NEB cat. S1509), as per the manufacturer’s instructions.

    Immunoprecipitation:

    Article Title: The E3 Ubiquitin Ligase TMEM129 Is a Tri-Spanning Transmembrane Protein
    Article Snippet: .. PNGase F: Eluted immunoprecipitated proteins were deglycosylated by adding G7 Reaction Buffer (New England Biolabs) and Remove-iT PNGase F (New England Biolabs), followed by incubation at 37 °C for 2 h. Note: the molecular weight of PNGase F (approximately 36 kDa) is approximately the same as that of TMEM129. .. To prevent distortion of the apparent molecular size of TMEM129 and putative glycosylated forms to occur, we used a removable PNGase F. Chitin Magnetic Beads (New England Biolabs) were pre-washed two times in TBS, added to the deglycosylated sample and incubated for 10 min at RT.

    Incubation:

    Article Title: Spontaneous Glycan Reattachment Following N-Glycanase Treatment of Influenza and HIV Vaccine Antigens.
    Article Snippet: .. In cells, asparagine/N-linked glycans are added to glycoproteins cotranslationally, in an attachment process that supports proper folding of the nascent polypeptide. .. In cells, asparagine/N-linked glycans are added to glycoproteins cotranslationally, in an attachment process that supports proper folding of the nascent polypeptide.

    Article Title: The Heterodimeric Glycoprotein Hormone, GPA2/GPB5, Regulates Ion Transport across the Hindgut of the Adult Mosquito, Aedes aegypti
    Article Snippet: .. Once denatured, the recombinant proteins were added to a deglycosylation reaction containing G7 Reaction buffer and 225 units of Remove-iT PNGase F (New England Biolabs, Whitby, ON) and incubated at 37°C for 1 hour. ..

    Article Title: The E3 Ubiquitin Ligase TMEM129 Is a Tri-Spanning Transmembrane Protein
    Article Snippet: .. PNGase F: Eluted immunoprecipitated proteins were deglycosylated by adding G7 Reaction Buffer (New England Biolabs) and Remove-iT PNGase F (New England Biolabs), followed by incubation at 37 °C for 2 h. Note: the molecular weight of PNGase F (approximately 36 kDa) is approximately the same as that of TMEM129. .. To prevent distortion of the apparent molecular size of TMEM129 and putative glycosylated forms to occur, we used a removable PNGase F. Chitin Magnetic Beads (New England Biolabs) were pre-washed two times in TBS, added to the deglycosylated sample and incubated for 10 min at RT.

    Article Title: Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens
    Article Snippet: .. Purified influenza H1 HA trimer was first de-N-glycosylated under non-denaturing conditions using commercial PNGase F tagged with a chitin-binding domain (CBD) (Remove-iT® PNGase F, Cat# P0706S, New England BioLabs) (=CBD-PNGase F) according to the manufacturer’s instructions (0.72 mg/mL HA, 40 000 units/mL CBD-PNGase F, 50 mM Na3 PO4 , pH 7.5; 24 h incubation at 37°C). .. Chitin magnetic beads (Cat# E8036S, New England BioLabs) equilibrated CBD binding buffer (500 mM NaCl, 20 mM Tris-HCI, 1 mM EDTA, pH 8) were incubated with the deglycosylation reaction (1 hr at 4°C, RotoFlex Plus Tube Rotator, Argos Technologies) and then applied to deplete CBD-PNGase from solution via separation on a magnetic column (NEB cat. S1509), as per the manufacturer’s instructions.

    Recombinant:

    Article Title: The Heterodimeric Glycoprotein Hormone, GPA2/GPB5, Regulates Ion Transport across the Hindgut of the Adult Mosquito, Aedes aegypti
    Article Snippet: .. Once denatured, the recombinant proteins were added to a deglycosylation reaction containing G7 Reaction buffer and 225 units of Remove-iT PNGase F (New England Biolabs, Whitby, ON) and incubated at 37°C for 1 hour. ..

    Molecular Weight:

    Article Title: The E3 Ubiquitin Ligase TMEM129 Is a Tri-Spanning Transmembrane Protein
    Article Snippet: .. PNGase F: Eluted immunoprecipitated proteins were deglycosylated by adding G7 Reaction Buffer (New England Biolabs) and Remove-iT PNGase F (New England Biolabs), followed by incubation at 37 °C for 2 h. Note: the molecular weight of PNGase F (approximately 36 kDa) is approximately the same as that of TMEM129. .. To prevent distortion of the apparent molecular size of TMEM129 and putative glycosylated forms to occur, we used a removable PNGase F. Chitin Magnetic Beads (New England Biolabs) were pre-washed two times in TBS, added to the deglycosylated sample and incubated for 10 min at RT.

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    New England Biolabs 12 tube magnetic separation rack
    12 Tube Magnetic Separation Rack, 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
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    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    12 tube magnetic separation rack - by Bioz Stars, 2020-08
    95/100 stars
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