To remove N-glycans, CD44 immunoprecipitates were treated with Peptide-N-Glycosidase (PNGase-F, New England Biolabs) according to the manufacturer’s instructions. Sialic acid residues were removed by treatment with 200 mU/mL Vibrio Cholerae sialidase (Roche Molecular Biochemicals) at 37°C for 1 hour. Cell surface proteins were cleaved with 0.1% bromelain (Sigma-Aldrich) for 1 hour at 37°C, and efficiency was assessed by flow cytometry staining for residual CD44 expression. For α(1,3)-exofucosylation, cells were treated with 0.07 mg/mL of fucosyltransferase VII (FTVII) (R&D systems) in HBSS buffer containing 10 mM HEPES, 0.1% human serum albumin and 1 mM GDP-fucose (Sigma-Aldrich) for 60 minutes at 37°C. As controls, cells were suspended in the same HBSS buffer but without addition of FTVII (buffer-treated).
Vibrio cholerae sialidase
Manufactured by Roche
Vibrio cholerae sialidase is a laboratory tool used to selectively cleave sialic acid residues from glycoconjugates. It functions as a carbohydrate-active enzyme, catalyzing the hydrolysis of terminal sialic acid residues from glycoproteins, glycolipids, and oligosaccharides.
Automatically generated - may contain errors
Lab products found in correlation
Gdp fucose, by Merck Group (1 mentions)
Bromelain, by Merck Group (1 mentions)
Optima l 80 xp, by Beckman Coulter (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
α2 3 n sialyltransferase, by Merck Group (1 mentions)
α2 6 n sialyltransferase, by Merck Group (1 mentions)
Streptavidin pe, by Southern Biotech (1 mentions)
4 protocols using vibrio cholerae sialidase
1
CD44 N-Glycan and Sialic Acid Removal
2
Purification and characterization of influenza VLPs
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HEK293T cells were transfected with pCMVdR8.91 (coding for HIV- Gag/Pol gene) and pcDNA-synH5 from a H5N1 known clinical isolate grown in the presence of soluble Vibrio cholerae sialidase (4 mU/mL; Roche). Supernatant was harvested 24 h post-transfection, filtered and concentrated on sucrose cushion (20% w/v of sucrose (Sigma, S-7903) filter sterilized on 0.45 um). After ultra-centrifugation (at 28,000 × g for 2.5 h on Optima L80 XP from Beckman Coulter equipped with rotor SW 32 TI), the pellet was re-suspended into 1/100th of the initial volume of the complete Dulbecco’s Modified Eagle’s Medium (DMEM/HIGH, Invitrogen #10569), 5% foetal bovine serum (FBS, Invitrogen #10500) and 1% Penicillin/ streptomycin (Invitrogen #15140). As the haemagglutinin construct contains a flag-tag on its C-terminal end, the content of HA in these VLP’s could be established by western-blot using anti-flag M2 peroxidase conjugate monoclonal antibodies (Sigma, A-8592) with a calibrating serial dilution of BAP-Flag protein (Sigma, P-7582).
3
Resialyated Turkey Red Blood Cell Hemagglutination Assay
4
Lectin Binding Analysis of hMSCs
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