β1 3 galactosidase  (New England Biolabs)

Journal: PLoS ONE

Article Title: New Tags for Recombinant Protein Detection and O-Glycosylation Reporters

doi: 10.1371/journal.pone.0096700

Figure Lengend Snippet: O-glycosylated tags. (A) WB of supernatants of 293T cells transfected with the reporter protein tagged with OG-tag, P-roTag or roTagO (previously indicated as 9–18, 11–21 and 9–21, respectively) treated (T) or not (Ctrl) with a glycosidase mix containing Neuraminidase, β1-3 Galactosidase and β-NAc-hexosaminidase. (B) WB of cellular extracts (E) and supernatants (S) of HEK 293T cells transfected with the reporter protein tagged with OG-tag and roTagO with or without the ER retention signal KDEL. In all panels blots were developed, as indicated, with anti-SV5 or anti-roTag/1F2.

Article Snippet: Where indicated samples were treated 2 hours at 37°C with a mix of Neuraminidase, β1-3 Galactosidase and β -N-Ac-hexosaminidase (New England Biolabs) in buffer 50 mM sodium citrate pH 4.5 according to manufacturer indications.

Techniques: Western Blot, Transfection

Journal: Anaerobe

Article Title: Release and utilization of N-acetyl-d-glucosamine from human milk oligosaccharides by Bifidobacterium longum subsp. infantis

doi: 10.1016/j.anaerobe.2012.04.012

Figure Lengend Snippet: Thin layer chromatography of co-incubations of B. infantis N-acetyl-β-D-hexosaminidases with LNT or LNH after treatment with β-galactosidases. Structures are illustrated below the figure. Lanes 1–8 and 26–28: standards (as indicated in the figure); lane 9: LNT with specific β1-3 galactosidase; lanes 10–12: LNT with a β1-3 galactosidase and Blon_0459, Blon_0732 or Blon_2355. Lane 13: LNH; lanes 14–16: LNH with either a β1-3, a β1-4 or both specific galactosidases; lanes 17–19: LNH with a β1-3 galactosidase and Blon_0459, Blon_0732 and Blon_2355, respectively; lanes 20–22: LNH with a β1-4 galactosidase and either Blon_0459, Blon_0732 and Blon_2355; lanes 23–25: LNH with both β1-3 and β1-4 galactosidase, as well as either Blon_0459, Blon_0732 and Blon_2355.

Article Snippet: Reactions were carried out for 60 min at 37 °C, and products were inactivated at 95 °C for 5 min. LNT and lacto-N-hexaose (LNH) at 1 mg/ml (0.5 μg final) were also coincubated with these enzymes, but with and without the addition of 10 units of β1-3 or β1-4 galactosidase, or both (New Englands Biolabs), for 1 h at 37 °C and pH 5.5.

Techniques: Thin Layer Chromatography

Journal: Scientific Reports

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

doi: 10.1038/s41598-018-35038-7

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

Article Snippet: In brief, glycans were digested with α2-3,6,8 Neuraminidase, α1-2,4,6 Fucosidase O, β1-4 Galactosidase, β-N -Acetyl-Glucosaminidase and α1-2,3,6 Mannnosidase (all from New England Biolabs) according to the manufacturer’s instructions.

Techniques: Infection, Hydrophilic Interaction Liquid Chromatography, Labeling

Journal: Scientific Reports

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

doi: 10.1038/s41598-018-35038-7

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

Article Snippet: In brief, glycans were digested with α2-3,6,8 Neuraminidase, α1-2,4,6 Fucosidase O, β1-4 Galactosidase, β-N -Acetyl-Glucosaminidase and α1-2,3,6 Mannnosidase (all from New England Biolabs) according to the manufacturer’s instructions.

Techniques: Infection

Journal: Cancers

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

doi: 10.3390/cancers10110460

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

Article Snippet: To generate sialic acids and galactose-free (asialyl-agalactosyl) IgG, normal serum IgG was treated with α2-3,6,8 neuraminidase and β1-4 galactosidase S (New England Biolabs, Ipswich, MA, USA) at 37 °C for 24 h. Normal serum IgG and asialyl-agalactosyl IgG were heated at 60 °C for 1 hour to generate aggregated IgG complexes.

Techniques: Whisker Assay, MANN-WHITNEY, Marker