Neuraminidase from clostridium perfringens

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Neuraminidase from Clostridium perfringens is an enzyme that catalyzes the removal of terminal sialic acid residues from glycoconjugates. It is commonly used in laboratory research applications.

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Lab products found in correlation

Fetuin, by Merck Group (2 mentions) Cell culture media, by Thermo Fisher Scientific (2 mentions) Dimethyl sulfoxide (dmso), by Merck Group (2 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions) Dtx880, by Beckman Coulter (1 mentions) Fc500 flow cytometer, by Beckman Coulter (1 mentions) Paraformaldehyde (pfa), by Merck Group (1 mentions) Versene solution, by Thermo Fisher Scientific (1 mentions) Ab18443, by Abcam (1 mentions) Fitc labeled human fibrinogen, by Innovative Research (1 mentions) Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions) 3fax peracetyl neu5ac, by Merck Group (1 mentions) Uea 1, by Vector Laboratories (1 mentions) Kaiser test, by AnaSpec (1 mentions) Pybop, by Chem-Impex (1 mentions) Fmoc nh peg 2 cooh 20 atoms, by Merck Group (1 mentions) Bsa conjugated monosaccharides, by Vector Laboratories (1 mentions) L fucose, by Vector Laboratories (1 mentions) D galactose, by Vector Laboratories (1 mentions) Streptomycin, by Merck Group (1 mentions)

Spelling variants of this product

Neuraminidase (91 citations) Clostridium perfringens (14 citations) Clostridium perfringens neuraminidase (12 citations) Neuraminidases from Clostridium perfringens (NanI) (2 citations)

14 protocols using neuraminidase from clostridium perfringens

Neuraminidase Treatment and Lectin Labeling

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Neuraminidase
from Clostridium perfringens (Sigma) was dissolved
in 1×
PBS, pH 7.4 at a final concentration of 10 U/mL. For neuraminidase
treatment CHO-K1_PD1-high cells were harvested using
enzyme free cell dissociation buffer and were resuspended in DMEM/F12
medium without FBS, P/S and G418. Neuraminidase treatment was performed
in a total volume of 150 μL with a cell concentration of 1 ×
10⁶ cells for 1h at 37 °C. After incubation cells
were centrifuged for 3 min at 1500×g and washed
1 time in labeling buffer. Subsequently, cells were labeled using
FITC-labeled lectin from Triticum vulgaris (Sigma)
at a concentration of 100 μg/mL for 30 min at 20 °C. The
labeled cells were centrifuged for 5 min at 1500×g, and the supernatant was removed. Directly prior to flow cytometry
or confocal microscopy, cells were resuspended in 200 μL of
labeling buffer.

Cremers G.A., Rosier B.J., Meijs A., Tito N.B., van Duijnhoven S.M., van Eenennaam H., Albertazzi L, & de Greef T.F. (2021). Determinants of Ligand-Functionalized DNA Nanostructure–Cell Interactions. Journal of the American Chemical Society, 143(27), 10131-10142.

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Preparation and Characterization of Modified IgA1 Complexes

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The purified monomeric IgA1 fractions from the recruited 17 healthy individuals were pooled and digested with neuraminidase from Clostridium perfringens (Sigma, St Louis, MO USA), and h-galactosidase from bovine testis (Sigma, St Louis, MO USA) to obtain de-sialic acid/de-galactose IgA1 molecules (ddIgA1) as described before [27 (link)]. In brief, 1 mg monomeric IgA1 were dissolved in 0.2 M sodium acetate buffer (pH 5.0), and added with 0.08 U neuraminidase and 0.02 U h-galactosidase to remove both sialic acid and Galactose. After incubation at 37 °C for 18 h, the ddIgA1 were dialyzed against PBS and concentrated using Vivaspin.
For in-vitro preparation of IgG-deglycosylated IgA1 complexes, a mixture of ddIgA1 and IgG (1:5 mass ratio) were incubated at 4 °C for 48 h, and then separated by gel filtration chromatography using Sephcrl S300 to obtain IgG-deglycosylated IgA1 complexes. The IgG-deglycosylated IgA1 complexes were also dialyzed against PBS and concentrated for cell culture experiments.
Meanwhile, ddIgA1 were separated by gel filtration chromatography using Sephcrl S300 to obtain monomeric ddIgA1 (mddIgA1) and polymeric ddIgA1 (pddIgA1), both of which were used as controls to treat cultured mesangial cells.

Zhao Y.F., Zhu L., Liu L.J., Shi S.F., Lv J.C, & Zhang H. (2017). Pathogenic role of glycan-specific IgG antibodies in IgA nephropathy. BMC Nephrology, 18, 301.

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Sialic Acid-Dependent Spike Protein Binding

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To analyze binding of the soluble spike proteins of the strains QX and B1648, we incubated the soluble proteins on cryosections of the oviduct at 4 °C for 1 h. To test if binding is sialic acid dependent we incubated the cryosections with 300 mU neuraminidase from Clostridium perfringens (Sigma-Aldrich, Steinheim, Germany) for 1 h at 37 °C before the incubation with the soluble proteins. Binding was detected with a secondary anti-human-Fc Cy-3 labeled antibody in a concentration of 1:750 (Sigma-Aldrich, Steinheim, Germany).

Mork A.K., Hesse M., Abd El Rahman S., Rautenschlein S., Herrler G, & Winter C. (2014). Differences in the tissue tropism to chicken oviduct epithelial cells between avian coronavirus IBV strains QX and B1648 are not related to the sialic acid binding properties of their spike proteins. Veterinary Research, 45(1), 67.

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MERS-CoV and HKU9-CoV Pseudovirus Entry

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Huh7 and RLK cells grown in 96-well plates were washed twice with PBS
(ThermoFisher Scientific) and incubated with neuraminidase from
Clostridium perfringens (Sigma) diluted in FBS-free growth
medium at 37 °C for 3 h. After the incubation, the cells were washed three
times and challenged with MERS–S- or HKU9–S-pseudoviruses, with or
without pre-incubation with the GRP78 polyclonal antibody (Abcam) for 1 h at 37
°C. Fresh complete medium with 10% FBS was replaced at 18 h post-infection.
Pseudovirus entry was quantified using a microplate reader (Beckman DTX880) as
RLU at 72 h post-infection.

Chu H., Chan C.M., Zhang X., Wang Y., Yuan S., Zhou J., Au-Yeung R.K., Sze K.H., Yang D., Shuai H., Hou Y., Li C., Zhao X., Poon V.K., Leung S.P., Yeung M.L., Yan J., Lu G., Jin D.Y., Gao G.F., Chan J.F, & Yuen K.Y. (2018). Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells. The Journal of Biological Chemistry, 293(30), 11709-11726.

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Quantification of Sialyl-Tn Expression

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Cells were detached using Versene solution (Thermo Fisher Scientific, Waltham, MA, USA), fixed with 2% paraformaldehyde (PFA; Sigma-Aldrich, St. Louis, MO, USA) and stained with mouse anti-TAG72 antibody [B72.3] (ab691; Abcam, Cambridge, UK) according with conditions previously described in Generation of an STn ESCC cell line topic. Mouse IgG1 [MOPC-21] isotype control (ab18443; Abcam, Cambridge, UK) was included as a negative control. In parallel, 10⁶ cells were digested with 70 mU neuraminidase from Clostridium perfringens (Sigma-Aldrich, St. Louis, MO, USA) in appropriate sodium acetate buffer at 37 °C overnight under mild agitation prior to STn staining, as a proper negative control. Data analysis was performed through CXP Software and results represent the standard deviation of three independent experiments performed in a FC500 Beckman Coulter flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN, USA).

Cotton S., Ferreira D., Soares J., Peixoto A., Relvas-Santos M., Azevedo R., Piairo P., Diéguez L., Palmeira C., Lima L., Silva A.M., Lara Santos L, & Ferreira J.A. (2021). Target Score—A Proteomics Data Selection Tool Applied to Esophageal Cancer Identifies GLUT1-Sialyl Tn Glycoforms as Biomarkers of Cancer Aggressiveness. International Journal of Molecular Sciences, 22(4), 1664.

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Analyzing Microglial CD11b Regulation

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All cell culture reagents were from Invitrogen (Paisley, UK). All chemicals were purchased from Sigma‐Aldrich (St. Louis, MO) unless indicated otherwise. Neuraminidase from Clostridium perfringens was from Sigma‐Aldrich. 3‐FAx‐peracetyl‐Neu5Ac was from Merck Millipore (Burlington, MA). Carboxylated Nile red 5 μm beads were from Spherotech (Chicago, IL). CD11b‐targeting, nontargeting siRNA, Lipofectamine 3000 reagent and mouse IgG2a isotype control were purchased from Thermo Scientific (Waltham, MA). Mouse monoclonal anti‐CD11b (clone OX‐42) antibody was from Serotec AbD (Hercules, CA). Recombinant human Tau protein (isoform 2N4R) was a kind gift from Dr Vilmante Borutaite. FITC‐labeled human fibrinogen was from Molecular Innovations (Novi, MI).

Allendorf D.H., Puigdellívol M, & Brown G.C. (2019). Activated microglia desialylate their surface, stimulating complement receptor 3‐mediated phagocytosis of neurons. Glia, 68(5), 989-998.

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Peptide Synthesis and Glycan Profiling

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TentaGel XV RAM resin was obtained from Rapp Polymer (Tuebingen, Germany). Fmoc-protected amino acids, 5(6)-carboxyfluorescein (5,6-FAM) and coupling reagents (HOBt, HBTU, PyBOP) were purchased from Chem-Impex (Wood Dale, IL) or Novabiochem (Gibbstown, NJ, USA). DIC was purchased from Acros Organics (Thermo Fisher Scientific, Waltham, MA). Fmoc-NH-(PEG)₂-COOH (20 atoms) was purchased from EMD Millipore (Billerica, MA). Kaiser test was purchased from AnaSpec (Fremont, CA). All solvents were purchased from Fisher Scientific (Atlanta, GA) or Sigma-Aldrich (St. Louis, MO), and were high-performance liquid chromatography (HPLC) grade. Fetuin, ASF, BME and iodine were purchased from Sigma-Aldrich (St. Louis, MO). Human cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA). Control antibiotics (penicillin and streptomycin) and enzymes (neuraminidase from Clostridium perfringens) were purchased from Sigma-Aldrich (St. Louis, MO). Cell culture media and PBS buffers were purchased from Fisher Scientific (Pittsburg, PA). FITC-labeled lectins (AAL, UEA-I, and SNA) and BSA-conjugated monosaccharides (L-fucose, D-glucose, D-galactose and L-galactose) were purchased from Vector Labs (Burlingame, CA). Cell Titer-Glo Luminescent Cell Viability Assay was purchased from Promega (Madison, WI).

Rodriguez M.C., Yongye A.B., Cudic M., Mayorga K.M., Liu E., Mueller B.M., Ainsley J., Karabencheva-Christova T., Christov C.Z., Cudic M, & Cudic P. (2017). Targeting cancer-specific glycans by cyclic peptide lectinomimics. Amino acids, 49(11), 1867-1883.

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Isolation and Characterization of Helicobacter bizzozeronii LPS

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LPS was extracted from biomass obtained after 48 h of incubation in BHI broth. Crude LPS was extracted by using the hot phenol-water method, and subsequent purification by enzymatic treatments (RNase A, DNase II and proteinase K) as described previously [6 (link)]. LPS were treated with Lysozyme to remove traces of peptidoglycan contamination [6 (link)]. After the enzymatic treatments, the LPS was precipitated at −20 °C overnight in 10 volumes of pure ethanol in presence of 0.03 M of sodium acetate and re-suspended in water and the concentration was then determined by purpald assay [20 (link)]. The LPS obtained was essentially free of proteins and nucleic acids, and it had an electrophoretic profile similar to that previously reported for the low-molecular-mass H. bizzozeronii LPS [11 (link)]. LPS was treated overnight with 6.7 U.mL⁻¹ of neuraminidase from Clostridium perfringens (Sigma-Aldrich) at pH 6. LPS neuraminidase treated and untreated samples were loaded on 15% TRIS-Glycine SDS-PAGE (Biorad, Hercules, CA, US), run for 2 h and 50 min at constant 20 mA and then silver stained as previous described [11 (link)].

Kondadi P.K., Revez J., Hänninen M.L, & Rossi M. (2015). Sialylation of Helicobacter bizzozeronii lipopolysaccharides modulates Toll-like receptor (TLR) 2 mediated response. Veterinary Research, 46(1), 4.

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Measuring 2,3-Linked Sialic Acid on Cell Surface

Cited 1 time

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For measuring the level of 2,3-linked sialic acid on the cell surface, cells were detached from plates using trypsin and stained with fluorescein-labeled MALI (Vector Laboratories). As a control, HeLa cells were treated with 100 milliunits/ml neuraminidase from Clostridium perfringens (Sigma-Aldrich) as described previously (10 (link)). Flow cytometry was performed using a BD Accuri flow cytometer (BD Biosciences), and the data were analyzed using the FlowJo program.

Kim H.S., Lee K., Bae S., Park J., Lee C.K., Kim M., Kim E., Kim M., Kim S., Kim C, & Kim J.S. (2017). CRISPR/Cas9-mediated gene knockout screens and target identification via whole-genome sequencing uncover host genes required for picornavirus infection. The Journal of Biological Chemistry, 292(25), 10664-10671.

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Isolation of Metabolites from S. pubescens

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Collection of the aerial part of S. pubescens was conducted at a local mountain in Chungcheongbuk-do, Republic of Korea in March 2022. At that time, S. pubescens was in the yellow flowers blooming growth stage. The collected plant was dried under dark conditions at room temperature for the isolation of the metabolites. Quercetin, methanol-d₄, chloroform-d, neuraminidase from Clostridium perfringens, 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid sodium salt hydrate, and dimethyl sulfoxide were purchased from Sigma Aldrich (St. Louis, MO, USA). Organic solvents, including hexane, chloroform, ethyl acetate, acetone, and methanol used for the separation, isolation, and purification of metabolites were purchased from Daejung Chemical and Metals (Siheung-si, Gyeonggi-do, Republic of Korea). Analytical-grade acetonitrile and water were purchased from Honeywell (Charlotte, NC, USA).

Son Y.G., Kim J.Y., Park J.Y., Kim K.D., Park K.H, & Kim J.Y. (2023). Inhibitory Potential of Quercetin Derivatives Isolated from the Aerial Parts of Siegesbeckia pubescens Makino against Bacterial Neuraminidase. Molecules, 28(14), 5365.

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