α1 2 3 mannosidase  (New England Biolabs)


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    Name:
    alpha 1 2 3 Mannosidase
    Description:
    alpha 1 2 3 Mannosidase 3 200 units
    Catalog Number:
    P0729L
    Price:
    518
    Category:
    Glycosidases
    Size:
    3 200 units
    Buy from Supplier


    Structured Review

    New England Biolabs α1 2 3 mannosidase
    alpha 1 2 3 Mannosidase
    alpha 1 2 3 Mannosidase 3 200 units
    https://www.bioz.com/result/α1 2 3 mannosidase/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    α1 2 3 mannosidase - by Bioz Stars, 2021-09
    93/100 stars

    Images

    1) Product Images from "Dectin-2 Recognizes Mannosylated O-antigens of Human Opportunistic Pathogens and Augments Lipopolysaccharide Activation of Myeloid Cells *"

    Article Title: Dectin-2 Recognizes Mannosylated O-antigens of Human Opportunistic Pathogens and Augments Lipopolysaccharide Activation of Myeloid Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.741256

    Dectin-2 recognizes H. alvei O-antigen. A–D , mouse Dectin-2 or mock BWZ cells were cultured for 16 h in the 96-well plate coated with purified LPS, plant-derived polysaccharides, lipid A, or nothing. The β-galactosidase activity was measured using the substrate. The data are expressed as the absorbance at 570 nm subtracted with the reference absorbance at 630 nm. E , the Man-LPS-coated plate was incubated with the α1-2,3-mannosidase at 37 °C for 14 h. The wells were washed with PBS, and the Dectin-2 reporter cells were added and analyzed as in A. F–H , BWZ cells expressing WT Dectin-2 or the QPD mutant were cultured in the presence of Man-LPS, E. coli O9a LPS, and the rough mutant LPS or 1.0 × 10 6 of PFA-fixed H. alvei PCM 1223. The binding was monitored as in A . Data are representative of three independent experiments with similar results. Error bars , S.D. Statistical analyses were performed by one-way ANOVA followed by Tukey's test ( A–D , G , and H ) or Student's t test ( E and F ). *, p
    Figure Legend Snippet: Dectin-2 recognizes H. alvei O-antigen. A–D , mouse Dectin-2 or mock BWZ cells were cultured for 16 h in the 96-well plate coated with purified LPS, plant-derived polysaccharides, lipid A, or nothing. The β-galactosidase activity was measured using the substrate. The data are expressed as the absorbance at 570 nm subtracted with the reference absorbance at 630 nm. E , the Man-LPS-coated plate was incubated with the α1-2,3-mannosidase at 37 °C for 14 h. The wells were washed with PBS, and the Dectin-2 reporter cells were added and analyzed as in A. F–H , BWZ cells expressing WT Dectin-2 or the QPD mutant were cultured in the presence of Man-LPS, E. coli O9a LPS, and the rough mutant LPS or 1.0 × 10 6 of PFA-fixed H. alvei PCM 1223. The binding was monitored as in A . Data are representative of three independent experiments with similar results. Error bars , S.D. Statistical analyses were performed by one-way ANOVA followed by Tukey's test ( A–D , G , and H ) or Student's t test ( E and F ). *, p

    Techniques Used: Cell Culture, Purification, Derivative Assay, Activity Assay, Incubation, Expressing, Mutagenesis, Binding Assay

    2) Product Images from "A library of chemically defined human N-glycans synthesized from microbial oligosaccharide precursors"

    Article Title: A library of chemically defined human N-glycans synthesized from microbial oligosaccharide precursors

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-15891-8

    Glycan synthesis strategies. Precursor glycans (1–3) were derived from ( a ) yeast invertase or ( b ) lipid-linked oligosaccharides (LLOs) from glycoengineered E. coli cells carrying plasmid pYCG, and subsequently used to synthesize glycans 4–21 . Enzymatic steps: (i) PNGase F (product shown is representative of high mannose yeast glycans); (ii) α1,2- and α1,6-mannosidase; (iii) jack bean α-mannosidase and α1,6-mannosidase; (iv) GnTI; (v) β1,4-galactosyltransferase; (vi) GnTIV; (vii) non-enzymatic hydrolysis of extracted LLOs; (viii) GnTII; (ix) GnTV; and (x) GnTIII.
    Figure Legend Snippet: Glycan synthesis strategies. Precursor glycans (1–3) were derived from ( a ) yeast invertase or ( b ) lipid-linked oligosaccharides (LLOs) from glycoengineered E. coli cells carrying plasmid pYCG, and subsequently used to synthesize glycans 4–21 . Enzymatic steps: (i) PNGase F (product shown is representative of high mannose yeast glycans); (ii) α1,2- and α1,6-mannosidase; (iii) jack bean α-mannosidase and α1,6-mannosidase; (iv) GnTI; (v) β1,4-galactosyltransferase; (vi) GnTIV; (vii) non-enzymatic hydrolysis of extracted LLOs; (viii) GnTII; (ix) GnTV; and (x) GnTIII.

    Techniques Used: Derivative Assay, Plasmid Preparation

    3) Product Images from "Identification and Functional Characterization of a Highly Divergent N-Acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei"

    Article Title: Identification and Functional Characterization of a Highly Divergent N-Acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.555029

    TbGT11-HA 3 expression and in vitro activity assay. A , Trypanosome bloodstream-form cell lysates from WT cells ( lane 1 ) or cells transfected with pLEW82-GT11-HA 3 ( lane 2 ) were separated by SDS-PAGE and analyzed by Western blotting with a rabbit anti-HA antibody. B, TLC autofluorography of in vitro reaction products. After incubation of TbGT11-HA 3 attached to anti-HA-conjugated magnetic beads with UDP-[ 3 H]GlcNAc as well as the acceptor substrates Man 3 GlcNAc 2 , Man 5 GlcNAc 2 , or no acceptor (H 2 O), reaction products were separated by TLC ( lanes 1–3 ). As a negative control, anti-HA magnetic beads incubated with lysates from cells not expressing TbGT11-HA 3 were used ( lane 4-6 ). C, the obtained [ 3 H]GlcNAcMan 3 GlcNAc 2 reaction product was separated by TLC before and after α1–2,3 mannosidase treatment and visualized by fluorography.
    Figure Legend Snippet: TbGT11-HA 3 expression and in vitro activity assay. A , Trypanosome bloodstream-form cell lysates from WT cells ( lane 1 ) or cells transfected with pLEW82-GT11-HA 3 ( lane 2 ) were separated by SDS-PAGE and analyzed by Western blotting with a rabbit anti-HA antibody. B, TLC autofluorography of in vitro reaction products. After incubation of TbGT11-HA 3 attached to anti-HA-conjugated magnetic beads with UDP-[ 3 H]GlcNAc as well as the acceptor substrates Man 3 GlcNAc 2 , Man 5 GlcNAc 2 , or no acceptor (H 2 O), reaction products were separated by TLC ( lanes 1–3 ). As a negative control, anti-HA magnetic beads incubated with lysates from cells not expressing TbGT11-HA 3 were used ( lane 4-6 ). C, the obtained [ 3 H]GlcNAcMan 3 GlcNAc 2 reaction product was separated by TLC before and after α1–2,3 mannosidase treatment and visualized by fluorography.

    Techniques Used: Expressing, In Vitro, Activity Assay, Transfection, SDS Page, Western Blot, Thin Layer Chromatography, Incubation, Magnetic Beads, Negative Control

    4) Product Images from "Assessing the Role of Pharyngeal Cell Surface Glycans in Group A Streptococcus Biofilm Formation"

    Article Title: Assessing the Role of Pharyngeal Cell Surface Glycans in Group A Streptococcus Biofilm Formation

    Journal: Antibiotics

    doi: 10.3390/antibiotics9110775

    Pre-treatment of pharyngeal cell surface with α1-6 mannosidase and Sialidase A results in significantly increased M12 GAS biofilm biomass. ( A ) Assay schematic for the characterization of biofilms formed on each of the exoglycosidase (α1-6 mannosidase, α1-2,3 mannosidase, and Sialidase (A) pre-treated pharyngeal monolayers vs. untreated. ( B ) Initial adherence enumerated for planktonic GAS upon 2 h incubation. 72 h biofilms are assessed for ( C ) biofilm biomass via crystal violet staining and ( D ) colony forming units via enumeration. Data represents mean ± SEM, with statistical analysis performed using a one-way ANOVA with Tukey’s multiple comparisons test * ( p ≤ 0.05); n = 3 biological replicates, with 3 technical replicates each.
    Figure Legend Snippet: Pre-treatment of pharyngeal cell surface with α1-6 mannosidase and Sialidase A results in significantly increased M12 GAS biofilm biomass. ( A ) Assay schematic for the characterization of biofilms formed on each of the exoglycosidase (α1-6 mannosidase, α1-2,3 mannosidase, and Sialidase (A) pre-treated pharyngeal monolayers vs. untreated. ( B ) Initial adherence enumerated for planktonic GAS upon 2 h incubation. 72 h biofilms are assessed for ( C ) biofilm biomass via crystal violet staining and ( D ) colony forming units via enumeration. Data represents mean ± SEM, with statistical analysis performed using a one-way ANOVA with Tukey’s multiple comparisons test * ( p ≤ 0.05); n = 3 biological replicates, with 3 technical replicates each.

    Techniques Used: Incubation, Staining

    Biofilm EPS increases significantly for biofilms formed on α1-6 mannosidase and Sialidase A pre-treated pharyngeal cell surfaces. ( A ) Assay schematic for the assessment of biofilm EPS resulting from biofilm formed on each of the exoglycosidase (α1-6 mannosidase, α1-2,3 mannosidase, and Sialidase A) pre-treated pharyngeal monolayers vs. untreated. 72 h biofilms were assessed for ( B ) EPS via DMMB staining of sulphated GAGs and ( C ) EPS associated components (eDNA and protein) via fluorescent staining with Sytox Blue and FilmTracer SYPRO Ruby biofilm matrix stain, respectively. Data represents mean ± SEM, with statistical analysis performed using a one-way ANOVA with Tukey’s multiple comparisons test * ( p ≤ 0.05) and ** ( p ≤ 0.01); n = 3 biological replicates, with 3 technical replicates each.
    Figure Legend Snippet: Biofilm EPS increases significantly for biofilms formed on α1-6 mannosidase and Sialidase A pre-treated pharyngeal cell surfaces. ( A ) Assay schematic for the assessment of biofilm EPS resulting from biofilm formed on each of the exoglycosidase (α1-6 mannosidase, α1-2,3 mannosidase, and Sialidase A) pre-treated pharyngeal monolayers vs. untreated. 72 h biofilms were assessed for ( B ) EPS via DMMB staining of sulphated GAGs and ( C ) EPS associated components (eDNA and protein) via fluorescent staining with Sytox Blue and FilmTracer SYPRO Ruby biofilm matrix stain, respectively. Data represents mean ± SEM, with statistical analysis performed using a one-way ANOVA with Tukey’s multiple comparisons test * ( p ≤ 0.05) and ** ( p ≤ 0.01); n = 3 biological replicates, with 3 technical replicates each.

    Techniques Used: Staining

    Visual inspection of 72 h M12 GAS biofilms captured via SEM revealed substantial EPS present in biofilms formed on exoglycosidase pre-treated pharyngeal cell monolayers. Images are representative of biofilms formed on ( A ) untreated, ( C ) α1-6 mannosidase, ( E ) α1-2,3 mannosidase, and ( G ) Sialidase A pre-treated pharyngeal monolayers. GAS biofilms show chained cocci (white arrows) arranged into three dimensional aggregated structures with EPS matrix material present (big and small black arrows). SEM images of ( B ) untreated, ( D ) α1-6 mannosidase, ( F ) α1-2,3 mannosidase, and ( H ) Sialidase A pre-treated Detroit 562 pharyngeal cell monolayers (without biofilm) are also included. Biofilms and Detroit 562 pharyngeal cell monolayers (without biofilm) were imaged using the JEOL JSM-7500 microscope at 15,000× and 500× magnification, respectively. SEM images were randomly selected and represent two b iological replicates with two technical replicates each.
    Figure Legend Snippet: Visual inspection of 72 h M12 GAS biofilms captured via SEM revealed substantial EPS present in biofilms formed on exoglycosidase pre-treated pharyngeal cell monolayers. Images are representative of biofilms formed on ( A ) untreated, ( C ) α1-6 mannosidase, ( E ) α1-2,3 mannosidase, and ( G ) Sialidase A pre-treated pharyngeal monolayers. GAS biofilms show chained cocci (white arrows) arranged into three dimensional aggregated structures with EPS matrix material present (big and small black arrows). SEM images of ( B ) untreated, ( D ) α1-6 mannosidase, ( F ) α1-2,3 mannosidase, and ( H ) Sialidase A pre-treated Detroit 562 pharyngeal cell monolayers (without biofilm) are also included. Biofilms and Detroit 562 pharyngeal cell monolayers (without biofilm) were imaged using the JEOL JSM-7500 microscope at 15,000× and 500× magnification, respectively. SEM images were randomly selected and represent two b iological replicates with two technical replicates each.

    Techniques Used: Microscopy

    Related Articles

    Incubation:

    Article Title: Dectin-2 Recognizes Mannosylated O-antigens of Human Opportunistic Pathogens and Augments Lipopolysaccharide Activation of Myeloid Cells *
    Article Snippet: .. Blocked wells were washed, and 50 units of α1–2,3 mannosidase (New England BioLabs) was added to the wells and incubated at 37 °C for 14 h. The reporter cells were added to test the binding. ..

    Article Title: Composition and Charge State Influence on the Ion-Neutral Collision Cross Sections of Protonated N-Linked Glycopeptides: An Experimental and Theoretical Deconstruction of Coulombic Repulsion vs. Charge Solvation Effects
    Article Snippet: .. Initially, 2 μL of 0.1 nmol/μL α(1→2,3) mannosidase was added to the glycopeptide preparation, followed by incubation at 37°C. ..

    Binding Assay:

    Article Title: Dectin-2 Recognizes Mannosylated O-antigens of Human Opportunistic Pathogens and Augments Lipopolysaccharide Activation of Myeloid Cells *
    Article Snippet: .. Blocked wells were washed, and 50 units of α1–2,3 mannosidase (New England BioLabs) was added to the wells and incubated at 37 °C for 14 h. The reporter cells were added to test the binding. ..

    Purification:

    Article Title: Assessing the Role of Pharyngeal Cell Surface Glycans in Group A Streptococcus Biofilm Formation
    Article Snippet: .. For mannosidases, the reaction volume comprises 3 µL 1 × GlycoBuffer 1 (NEB, Notting Hill, Australia), 0.3 µL 100 µg/mL purified BSA (NEB, Notting Hill, Australia), 0.2 µL α1-6 mannosidase (8 U) or α1-2,3 mannosidase (8 U) (NEB, Notting Hill, Australia), and 26.5 µL PBS. ..

    other:

    Article Title: Reconstitution of the lipid-linked oligosaccharide pathway for assembly of high-mannose N-glycans
    Article Snippet: Mannosidase digestions Digestion of glycans (2.5 nmol) with 3.2 U of α1,2-3-mannosidase (Xanthomonas manihotis , New England Biolabs, MA, USA), 4 U of α1,6-mannosidase (X. manihotis , New England Biolabs), and 0.1 mU of α1,2-mannosidase (Aspergillus saitoi , ProZyme, CA, USA) were performed in 10 μL at 25 °C for 16 h in buffers supplied by the manufacture.

    Article Title: XBP1s Links the Unfolded Protein Response to the Molecular Architecture of Mature N-Glycans
    Article Snippet: PNGase-F, Endo-H, Endo-F3 , α1–2,3-mannosidase, and α1–6-mannosidase enzymes were obtained from NEB.

    Mass Spectrometry:

    Article Title: Comparison of RP-HPLC modes to analyse the N-glycome of the free-living nematode Pristionchus pacificus
    Article Snippet: .. Further analysis by MALDI-TOF MS was performed after treatment overnight with either β-galactosidase (recombinant Aspergillus niger lacA; prepared in-house ( )), α-fucosidase (bovine kidney from Sigma-Aldrich), α-mannosidase ( Xanthomonas manihotis α1,2/3-specific from NEB) or β- N -acetylhexosaminidase (recombinant Apis mellifera FDL; prepared in-house, specific for the N- acetylglucosamine attached to core α1,3-mannose, i.e., the residue transferred by GlcNAc-TI ( )) in 25 mM ammonium acetate, pH 4.5, at 37 °C. ..

    Recombinant:

    Article Title: Comparison of RP-HPLC modes to analyse the N-glycome of the free-living nematode Pristionchus pacificus
    Article Snippet: .. Further analysis by MALDI-TOF MS was performed after treatment overnight with either β-galactosidase (recombinant Aspergillus niger lacA; prepared in-house ( )), α-fucosidase (bovine kidney from Sigma-Aldrich), α-mannosidase ( Xanthomonas manihotis α1,2/3-specific from NEB) or β- N -acetylhexosaminidase (recombinant Apis mellifera FDL; prepared in-house, specific for the N- acetylglucosamine attached to core α1,3-mannose, i.e., the residue transferred by GlcNAc-TI ( )) in 25 mM ammonium acetate, pH 4.5, at 37 °C. ..

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  • 93
    New England Biolabs α1 2 3 mannosidase
    Dectin-2 recognizes H. alvei O-antigen. A–D , mouse Dectin-2 or mock BWZ cells were cultured for 16 h in the 96-well plate coated with purified LPS, plant-derived polysaccharides, lipid A, or nothing. The β-galactosidase activity was measured using the substrate. The data are expressed as the absorbance at 570 nm subtracted with the reference absorbance at 630 nm. E , the Man-LPS-coated plate was incubated with the <t>α1-2,3-mannosidase</t> at 37 °C for 14 h. The wells were washed with PBS, and the Dectin-2 reporter cells were added and analyzed as in A. F–H , BWZ cells expressing WT Dectin-2 or the QPD mutant were cultured in the presence of Man-LPS, E. coli O9a LPS, and the rough mutant LPS or 1.0 × 10 6 of PFA-fixed H. alvei PCM 1223. The binding was monitored as in A . Data are representative of three independent experiments with similar results. Error bars , S.D. Statistical analyses were performed by one-way ANOVA followed by Tukey's test ( A–D , G , and H ) or Student's t test ( E and F ). *, p
    α1 2 3 Mannosidase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α1 2 3 mannosidase/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    α1 2 3 mannosidase - by Bioz Stars, 2021-09
    93/100 stars
      Buy from Supplier

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    Dectin-2 recognizes H. alvei O-antigen. A–D , mouse Dectin-2 or mock BWZ cells were cultured for 16 h in the 96-well plate coated with purified LPS, plant-derived polysaccharides, lipid A, or nothing. The β-galactosidase activity was measured using the substrate. The data are expressed as the absorbance at 570 nm subtracted with the reference absorbance at 630 nm. E , the Man-LPS-coated plate was incubated with the α1-2,3-mannosidase at 37 °C for 14 h. The wells were washed with PBS, and the Dectin-2 reporter cells were added and analyzed as in A. F–H , BWZ cells expressing WT Dectin-2 or the QPD mutant were cultured in the presence of Man-LPS, E. coli O9a LPS, and the rough mutant LPS or 1.0 × 10 6 of PFA-fixed H. alvei PCM 1223. The binding was monitored as in A . Data are representative of three independent experiments with similar results. Error bars , S.D. Statistical analyses were performed by one-way ANOVA followed by Tukey's test ( A–D , G , and H ) or Student's t test ( E and F ). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Dectin-2 Recognizes Mannosylated O-antigens of Human Opportunistic Pathogens and Augments Lipopolysaccharide Activation of Myeloid Cells *

    doi: 10.1074/jbc.M116.741256

    Figure Lengend Snippet: Dectin-2 recognizes H. alvei O-antigen. A–D , mouse Dectin-2 or mock BWZ cells were cultured for 16 h in the 96-well plate coated with purified LPS, plant-derived polysaccharides, lipid A, or nothing. The β-galactosidase activity was measured using the substrate. The data are expressed as the absorbance at 570 nm subtracted with the reference absorbance at 630 nm. E , the Man-LPS-coated plate was incubated with the α1-2,3-mannosidase at 37 °C for 14 h. The wells were washed with PBS, and the Dectin-2 reporter cells were added and analyzed as in A. F–H , BWZ cells expressing WT Dectin-2 or the QPD mutant were cultured in the presence of Man-LPS, E. coli O9a LPS, and the rough mutant LPS or 1.0 × 10 6 of PFA-fixed H. alvei PCM 1223. The binding was monitored as in A . Data are representative of three independent experiments with similar results. Error bars , S.D. Statistical analyses were performed by one-way ANOVA followed by Tukey's test ( A–D , G , and H ) or Student's t test ( E and F ). *, p

    Article Snippet: Blocked wells were washed, and 50 units of α1–2,3 mannosidase (New England BioLabs) was added to the wells and incubated at 37 °C for 14 h. The reporter cells were added to test the binding.

    Techniques: Cell Culture, Purification, Derivative Assay, Activity Assay, Incubation, Expressing, Mutagenesis, Binding Assay

    Glycan synthesis strategies. Precursor glycans (1–3) were derived from ( a ) yeast invertase or ( b ) lipid-linked oligosaccharides (LLOs) from glycoengineered E. coli cells carrying plasmid pYCG, and subsequently used to synthesize glycans 4–21 . Enzymatic steps: (i) PNGase F (product shown is representative of high mannose yeast glycans); (ii) α1,2- and α1,6-mannosidase; (iii) jack bean α-mannosidase and α1,6-mannosidase; (iv) GnTI; (v) β1,4-galactosyltransferase; (vi) GnTIV; (vii) non-enzymatic hydrolysis of extracted LLOs; (viii) GnTII; (ix) GnTV; and (x) GnTIII.

    Journal: Scientific Reports

    Article Title: A library of chemically defined human N-glycans synthesized from microbial oligosaccharide precursors

    doi: 10.1038/s41598-017-15891-8

    Figure Lengend Snippet: Glycan synthesis strategies. Precursor glycans (1–3) were derived from ( a ) yeast invertase or ( b ) lipid-linked oligosaccharides (LLOs) from glycoengineered E. coli cells carrying plasmid pYCG, and subsequently used to synthesize glycans 4–21 . Enzymatic steps: (i) PNGase F (product shown is representative of high mannose yeast glycans); (ii) α1,2- and α1,6-mannosidase; (iii) jack bean α-mannosidase and α1,6-mannosidase; (iv) GnTI; (v) β1,4-galactosyltransferase; (vi) GnTIV; (vii) non-enzymatic hydrolysis of extracted LLOs; (viii) GnTII; (ix) GnTV; and (x) GnTIII.

    Article Snippet: The resulting high-mannose precursor oligosaccharides were specifically trimmed with both X. manihotis α1-6-mannosidase (ProZyme) and A. saitoi α1-2-mannosidase (New England Biolabs) to produce the human-type Man5 GlcNAc2 glycan, since we empirically determined that there were no terminal α(1,3)-mannose residues.

    Techniques: Derivative Assay, Plasmid Preparation

    TbGT11-HA 3 expression and in vitro activity assay. A , Trypanosome bloodstream-form cell lysates from WT cells ( lane 1 ) or cells transfected with pLEW82-GT11-HA 3 ( lane 2 ) were separated by SDS-PAGE and analyzed by Western blotting with a rabbit anti-HA antibody. B, TLC autofluorography of in vitro reaction products. After incubation of TbGT11-HA 3 attached to anti-HA-conjugated magnetic beads with UDP-[ 3 H]GlcNAc as well as the acceptor substrates Man 3 GlcNAc 2 , Man 5 GlcNAc 2 , or no acceptor (H 2 O), reaction products were separated by TLC ( lanes 1–3 ). As a negative control, anti-HA magnetic beads incubated with lysates from cells not expressing TbGT11-HA 3 were used ( lane 4-6 ). C, the obtained [ 3 H]GlcNAcMan 3 GlcNAc 2 reaction product was separated by TLC before and after α1–2,3 mannosidase treatment and visualized by fluorography.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification and Functional Characterization of a Highly Divergent N-Acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei

    doi: 10.1074/jbc.M114.555029

    Figure Lengend Snippet: TbGT11-HA 3 expression and in vitro activity assay. A , Trypanosome bloodstream-form cell lysates from WT cells ( lane 1 ) or cells transfected with pLEW82-GT11-HA 3 ( lane 2 ) were separated by SDS-PAGE and analyzed by Western blotting with a rabbit anti-HA antibody. B, TLC autofluorography of in vitro reaction products. After incubation of TbGT11-HA 3 attached to anti-HA-conjugated magnetic beads with UDP-[ 3 H]GlcNAc as well as the acceptor substrates Man 3 GlcNAc 2 , Man 5 GlcNAc 2 , or no acceptor (H 2 O), reaction products were separated by TLC ( lanes 1–3 ). As a negative control, anti-HA magnetic beads incubated with lysates from cells not expressing TbGT11-HA 3 were used ( lane 4-6 ). C, the obtained [ 3 H]GlcNAcMan 3 GlcNAc 2 reaction product was separated by TLC before and after α1–2,3 mannosidase treatment and visualized by fluorography.

    Article Snippet: For product analysis, samples were treated with α1–2,3-mannosidase from Xanthomonas manihotis (New England Biolabs) before TLC analysis.

    Techniques: Expressing, In Vitro, Activity Assay, Transfection, SDS Page, Western Blot, Thin Layer Chromatography, Incubation, Magnetic Beads, Negative Control

    Pre-treatment of pharyngeal cell surface with α1-6 mannosidase and Sialidase A results in significantly increased M12 GAS biofilm biomass. ( A ) Assay schematic for the characterization of biofilms formed on each of the exoglycosidase (α1-6 mannosidase, α1-2,3 mannosidase, and Sialidase (A) pre-treated pharyngeal monolayers vs. untreated. ( B ) Initial adherence enumerated for planktonic GAS upon 2 h incubation. 72 h biofilms are assessed for ( C ) biofilm biomass via crystal violet staining and ( D ) colony forming units via enumeration. Data represents mean ± SEM, with statistical analysis performed using a one-way ANOVA with Tukey’s multiple comparisons test * ( p ≤ 0.05); n = 3 biological replicates, with 3 technical replicates each.

    Journal: Antibiotics

    Article Title: Assessing the Role of Pharyngeal Cell Surface Glycans in Group A Streptococcus Biofilm Formation

    doi: 10.3390/antibiotics9110775

    Figure Lengend Snippet: Pre-treatment of pharyngeal cell surface with α1-6 mannosidase and Sialidase A results in significantly increased M12 GAS biofilm biomass. ( A ) Assay schematic for the characterization of biofilms formed on each of the exoglycosidase (α1-6 mannosidase, α1-2,3 mannosidase, and Sialidase (A) pre-treated pharyngeal monolayers vs. untreated. ( B ) Initial adherence enumerated for planktonic GAS upon 2 h incubation. 72 h biofilms are assessed for ( C ) biofilm biomass via crystal violet staining and ( D ) colony forming units via enumeration. Data represents mean ± SEM, with statistical analysis performed using a one-way ANOVA with Tukey’s multiple comparisons test * ( p ≤ 0.05); n = 3 biological replicates, with 3 technical replicates each.

    Article Snippet: For mannosidases, the reaction volume comprises 3 µL 1 × GlycoBuffer 1 (NEB, Notting Hill, Australia), 0.3 µL 100 µg/mL purified BSA (NEB, Notting Hill, Australia), 0.2 µL α1-6 mannosidase (8 U) or α1-2,3 mannosidase (8 U) (NEB, Notting Hill, Australia), and 26.5 µL PBS.

    Techniques: Incubation, Staining

    Biofilm EPS increases significantly for biofilms formed on α1-6 mannosidase and Sialidase A pre-treated pharyngeal cell surfaces. ( A ) Assay schematic for the assessment of biofilm EPS resulting from biofilm formed on each of the exoglycosidase (α1-6 mannosidase, α1-2,3 mannosidase, and Sialidase A) pre-treated pharyngeal monolayers vs. untreated. 72 h biofilms were assessed for ( B ) EPS via DMMB staining of sulphated GAGs and ( C ) EPS associated components (eDNA and protein) via fluorescent staining with Sytox Blue and FilmTracer SYPRO Ruby biofilm matrix stain, respectively. Data represents mean ± SEM, with statistical analysis performed using a one-way ANOVA with Tukey’s multiple comparisons test * ( p ≤ 0.05) and ** ( p ≤ 0.01); n = 3 biological replicates, with 3 technical replicates each.

    Journal: Antibiotics

    Article Title: Assessing the Role of Pharyngeal Cell Surface Glycans in Group A Streptococcus Biofilm Formation

    doi: 10.3390/antibiotics9110775

    Figure Lengend Snippet: Biofilm EPS increases significantly for biofilms formed on α1-6 mannosidase and Sialidase A pre-treated pharyngeal cell surfaces. ( A ) Assay schematic for the assessment of biofilm EPS resulting from biofilm formed on each of the exoglycosidase (α1-6 mannosidase, α1-2,3 mannosidase, and Sialidase A) pre-treated pharyngeal monolayers vs. untreated. 72 h biofilms were assessed for ( B ) EPS via DMMB staining of sulphated GAGs and ( C ) EPS associated components (eDNA and protein) via fluorescent staining with Sytox Blue and FilmTracer SYPRO Ruby biofilm matrix stain, respectively. Data represents mean ± SEM, with statistical analysis performed using a one-way ANOVA with Tukey’s multiple comparisons test * ( p ≤ 0.05) and ** ( p ≤ 0.01); n = 3 biological replicates, with 3 technical replicates each.

    Article Snippet: For mannosidases, the reaction volume comprises 3 µL 1 × GlycoBuffer 1 (NEB, Notting Hill, Australia), 0.3 µL 100 µg/mL purified BSA (NEB, Notting Hill, Australia), 0.2 µL α1-6 mannosidase (8 U) or α1-2,3 mannosidase (8 U) (NEB, Notting Hill, Australia), and 26.5 µL PBS.

    Techniques: Staining

    Visual inspection of 72 h M12 GAS biofilms captured via SEM revealed substantial EPS present in biofilms formed on exoglycosidase pre-treated pharyngeal cell monolayers. Images are representative of biofilms formed on ( A ) untreated, ( C ) α1-6 mannosidase, ( E ) α1-2,3 mannosidase, and ( G ) Sialidase A pre-treated pharyngeal monolayers. GAS biofilms show chained cocci (white arrows) arranged into three dimensional aggregated structures with EPS matrix material present (big and small black arrows). SEM images of ( B ) untreated, ( D ) α1-6 mannosidase, ( F ) α1-2,3 mannosidase, and ( H ) Sialidase A pre-treated Detroit 562 pharyngeal cell monolayers (without biofilm) are also included. Biofilms and Detroit 562 pharyngeal cell monolayers (without biofilm) were imaged using the JEOL JSM-7500 microscope at 15,000× and 500× magnification, respectively. SEM images were randomly selected and represent two b iological replicates with two technical replicates each.

    Journal: Antibiotics

    Article Title: Assessing the Role of Pharyngeal Cell Surface Glycans in Group A Streptococcus Biofilm Formation

    doi: 10.3390/antibiotics9110775

    Figure Lengend Snippet: Visual inspection of 72 h M12 GAS biofilms captured via SEM revealed substantial EPS present in biofilms formed on exoglycosidase pre-treated pharyngeal cell monolayers. Images are representative of biofilms formed on ( A ) untreated, ( C ) α1-6 mannosidase, ( E ) α1-2,3 mannosidase, and ( G ) Sialidase A pre-treated pharyngeal monolayers. GAS biofilms show chained cocci (white arrows) arranged into three dimensional aggregated structures with EPS matrix material present (big and small black arrows). SEM images of ( B ) untreated, ( D ) α1-6 mannosidase, ( F ) α1-2,3 mannosidase, and ( H ) Sialidase A pre-treated Detroit 562 pharyngeal cell monolayers (without biofilm) are also included. Biofilms and Detroit 562 pharyngeal cell monolayers (without biofilm) were imaged using the JEOL JSM-7500 microscope at 15,000× and 500× magnification, respectively. SEM images were randomly selected and represent two b iological replicates with two technical replicates each.

    Article Snippet: For mannosidases, the reaction volume comprises 3 µL 1 × GlycoBuffer 1 (NEB, Notting Hill, Australia), 0.3 µL 100 µg/mL purified BSA (NEB, Notting Hill, Australia), 0.2 µL α1-6 mannosidase (8 U) or α1-2,3 mannosidase (8 U) (NEB, Notting Hill, Australia), and 26.5 µL PBS.

    Techniques: Microscopy