In order to expose N-glycans to Fbs1, N-glycoproteins were first denatured. RNase B or human IgG was denatured by boiling in 0.5% SDS and 40 mM DTT. After boiling, 1% NP-40 was added to counteract the effect of SDS. The denatured RNase B or human IgG was either untreated or treated with PNGase F (for 1 h at 37 °C in 50 mM sodium phosphate, pH 7.5) to remove N-glycans as indicated in the figures. The mixture of RNase B and fetuin was denatured by boiling for 10 min in the presence of 1 × Rapid PNGase F buffer (NEB). The denatured proteins were incubated with Fbs1 beads at 4 °C for 1 h. After incubation, the beads were washed, and the bound proteins on the beads were eluted in 1 × SDS gel loading buffer and analysed by SDS–PAGE/Coomassie blue staining. ImageJ (National Institutes of Health) was used to quantify the protein bands.
Rapid pngase f buffer
Manufactured by New England Biolabs
Sourced in United States
Rapid PNGase F buffer is a solution designed to facilitate the deglycosylation of glycoproteins using the PNGase F enzyme. It provides the necessary conditions for the efficient removal of N-linked glycans from protein samples.
Automatically generated - may contain errors
4 protocols using rapid pngase f buffer
1
Deglycosylation of Proteins for Fbs1 Binding
2
Glycan Analysis of Human Alpha-1-Acid Glycoprotein
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All chemicals and reagents were at least
analytical reagent grade and used without further purification. Rapid
PNGase F and Rapid PNGase F Buffer were supplied by New England Biolabs
(Ipswich, USA). Human alpha-1-acid glycoprotein (hAGP, ≥95%),
Discovery Glycan solid phase extraction (SPE) tubes, TFA (≥99%),
procainamide hydrochlorid (≥98%), and both trisaccharide standards
6′/3′-Sialyl-N-acetyllactosamine were
purchased from Sigma-Aldrich (St. Louis, USA). The fucosylated standard
3′-Sialyl-Lewis X was supplied by Biosynth Carbosynth (UK).
Hypercarb SPE tubes were purchased from Thermo Fisher Scientific (Waltham,
USA). Ammonium formate (>99%) was obtained from VWR International
(Radnor, USA). All solvents (acetonitrile, methanol, and water) were
LC-MS grade and purchased from Sigma-Aldrich (St. Louis, USA).
analytical reagent grade and used without further purification. Rapid
PNGase F and Rapid PNGase F Buffer were supplied by New England Biolabs
(Ipswich, USA). Human alpha-1-acid glycoprotein (hAGP, ≥95%),
Discovery Glycan solid phase extraction (SPE) tubes, TFA (≥99%),
procainamide hydrochlorid (≥98%), and both trisaccharide standards
6′/3′-Sialyl-N-acetyllactosamine were
purchased from Sigma-Aldrich (St. Louis, USA). The fucosylated standard
3′-Sialyl-Lewis X was supplied by Biosynth Carbosynth (UK).
Hypercarb SPE tubes were purchased from Thermo Fisher Scientific (Waltham,
USA). Ammonium formate (>99%) was obtained from VWR International
(Radnor, USA). All solvents (acetonitrile, methanol, and water) were
LC-MS grade and purchased from Sigma-Aldrich (St. Louis, USA).
3
Rapid PNGase F-Mediated Glycan Release
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All chemicals and reagents were at least analytical reagent grade and used without further purification. Rapid™ PNGase F and Rapid™ PNGase F Buffer were supplied by New England Biolabs (Ipswich, USA). Immunoglobulin from human serum (hIgG, ≥ 95%), human alpha-1-acid glycoprotein (hAGP, ≥ 95%), dextran Mw5000, dextran Mw1000, Discovery Glycan solid-phase extraction (SPE) tubes, and procainamide hydrochloride (≥ 98%) were purchased from Sigma-Aldrich (St. Louis, USA). Ammonium formate (> 99%) was obtained from VWR International (Radnor, USA). All solvents (acetonitrile, water) were LC–MS grade and purchased from Sigma-Aldrich (St. Louis, USA).
4
IgG N-Glycan Purification and Desalting
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Samples after lyophilization were resuspended in 19 µl of Rapid PNGase F buffer (New England Biolabs). They were then denatured for 15 min at 80°C. After cooling the plate to RT, 0.9 µl of Rapid PNGase F (New England Biolabs, P0710S) was added to each well and the plate was incubated for 25 min at 50°C. After deglycosylation, samples were diluted with 100 µl Milli-Q. IgG-released N-glycans were desalted by solid-phase extraction on HyperSepTM Hypercarb™ SPE 96-Well Plates with 10 mg bed weight (Thermo Fisher Scientific, 60302-606). The plate was prewashed three times with 400 µl of 80% acetonitrile (AcN) with 0.1% trifluoroacetic acid (TFA) and then three times with 400 µl of Milli-Q, on the vacuum filtration manifold. The samples were then applied and the columns were washed three times with 400 µl of Milli-Q. In the next step, N-oligosaccharides were eluted with 25% AcN + 0.05% TFA into a 96-well Sample Collection Plate (Waters, 186005837). The collected fractions were dried-down by lyophilization (Labconco).
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