New England Biolabs, P6042S, Fetuin

Fetuin is a glycoprotein containing sialylated N-linked and O-linked glycans that can be used as a positive control for endoglycosidase enzymes that cleave both N-linked and O-linked carbohydrates. Related Categories Glycoprotein Substrates Applications Expression Systems,, Glycan Sequencing,, Recombinant Glycoprotein Expression, Specification Storage Buffer 1X Milli-Q water Molecular Weight Apparent: 48.4 kDa FAQ Q: What is a good endoglycosidase control substrate? A: For O-Glycosidase (NEB# P0733) and the Protein Deglycosylation Mix (NEB# P6039S), we suggest Fetuin (NEB# P6042). For PNGase F (NEB # P0704, P0705, P0706) we suggest RNase B (NEB# P7817S) or Fetuin (NEB# P6042). Q: Why is Fetuin a good control substrate for both O-Glycosidase, PNGase F and the Protein Deglycosylation Mix? A: Fetuin is a glycoprotein that contains both sialylated N-linked and O-linked glycans and can be analyzed by SDS-PAGE gel shift analysis, mass spectrometry or other various methods Q: What is a typical Fetuin deglycosylation reaction condition? A: Combine 2 μl of Fetuin, 1 μl of 10X Glycoprotein Denaturing Buffer, and 5 μl of H2O. Incubate at 95°C for 10 minutes. Add 1 μl of 10X GlycoBuffer 2 and 1 μl of 10% NP-40. Add 1 μl of endoglycosidase. Incubate at 37°C for 1 hour. Visualize reaction products on 10–20% SDS-PAGE gel. Q: Do detergents inhibit exoglycosidases/endoglycosidases? A: At moderate levels (0.5-1.0% ionic and non-ionic detergents) most of the glycosidases show satisfactory activity or are unaffected. Exceptions are: PNGase F (all formulations), O-glycosidase, and β-N-Acetylglucosaminidase, which are inhibited by SDS. It is imperative to add NP-40 to a final concentration of 1% to your reaction mixture in order to counteract detergent inhibition. Endo D is also inhibited by SDS, however NP-40 does not counteract this inhibition. Finally, common stabilizing reagents such as Tween, Triton X-100, NP-40, octyl glucoside and non-detergent sulfobetaines, as well as traces of organic solvents, can prevent optimal rapid deglycosylation by Rapid PNGase F. Q: How much endoglycosidase should I use to deglycosylate Fetuin under native conditions? A: When Fetuin is not denatured the endoglycosidase may have a more difficult time reaching the cleavage site of the carbohydrate (because of the secondary and tertiary protein structure). Extra enzyme and extended incubation times will help but these values are specific to each protein and must be determined empirically.