Enzymatic DeGlycoMx Kit
This kit developed for protein deglycosylation includes our DeGlycoMx, a premixed cocktail of the enzymes required to remove all N-linked oligosaccharides and most O-linked sugars from 0.5 mg of glycoprotein, via 10 reactions of up to 50 micrograms of protein per reaction.
The kit is easy-to-use and effective: add 2 µl of the DeGlycoMx enzyme to your denatured sample and the included buffer and incubate for 3 hours at 37˚C.
KE-DGMX – 20 microliters
KE-DGMX-100 – 100 microliters
The enzymes are premixed in either one 20 or 100 microliter vial. This allows researchers to quickly remove most all glycans (with the exception of O-Linked mucins) from their glycoprotein in both denatured and native conditions. By combining the enzymes into one easy-to-use premixed solution, researchers save time and money in the pursuit of protein deglycosylation. The enzymes can also be supplied individually in 20 microliter vials of each enzyme (KE-DG01) allowing the researcher to the flexibility to characterize the glycans attached to their glycoprotein more fully than with our mixture of enzymes.
Includes one 20 microlitre vial including the following enzymes in solution:
- PNGase F (Chryseobacterium meningosepticum)
- O-Glycosidase (Streptococcus pneumoniae)
- Neuraminidase (Arthrobacter ureafaciens)
- Beta-Galactosidase (Streptococcus pneumoniae)
- β-N-Acetylglucosaminidase (Streptococcus pneumonia)
Other Supplied Reagents:
- Reaction buffer
- Denaturation Solution
- Triton X
The Enzymatic DeGlycoMx Kit will remove all N-linked oligosaccharides and many O-linked oligosaccharides from glycoproteins. Protein deglycosylation for N-linked glycans (Asparganine-linked) is performed using the enzyme PNGase F. In addition, all Serine or Threonine linked (O-linked) Gal-(b1-3)-GalNAc-(a1) and all sialic acid substituted Gal-(b1-3)-GalNAc-(a1) will be removed using the combination of Neuraminidase and O-Glycosidase. The addition of Beta-Galactosidase and β-N-acetylglucosaminidase will assist in the deglycosylation of larger O-link structures.
1. Mix 10 µl of reaction buffer with up to 50 µg of glycoprotein in 33 µl distilled water in a 1.5 µl tube.
2. Add 2.5 µl denaturation solution. Mix gently and place in boiling water bath for 5 minutes. Chill on ice.
3. Add 2.5 µl of Triton-X.
4. Add 2 µls of the DeGlycoMx enzyme cocktail. Incubate for 3 hours at 37˚C.
Note: Denaturation increases the rate of enzyme digestion up to 10 fold. If denaturation is not desired omit step 2-3, add with 5 µl of distilled water and increase incubation time up to 24 hours.
References for Protein Deglycosylation
Kobata, A. Use of endo- and exoglycosidases for structural studies of glycoconjugates. Anal Biochem 100:1- 14 (1979).
Kim MS, Leahy D. Enzymatic deglycosylation of glycoproteins. Methods Enzymol.533:259-63 (2013).
Magnelli PE, Bielik AM, Guthrie EP. Identification and characterization of protein glycosylation using specific endo- and exoglycosidases. J Vis Exp. Dec 26;(58):e3749 (2011).
Segu ZM, Hussein A, Novotny MV, Mechref Y. Assigning N-glycosylation sites of glycoproteins using LC/MSMS in conjunction with endo-M/exoglycosidase mixture. J Proteome Res. Jul 2;9(7):3598-607 (2010).
Sojar, H. T. and O.P. Bahl. A chemical method for the deglycosylation of proteins. Arch Biochem Biophys 259:52-57 (1987).
Tarentino A.L. and T.H. Plummer. Enzymatic deglycosylation of asparagine- linked glycans: purification, properties, and specificitly of oligosaccharide- cleaving enzymes from Flavobacterium meningosepticum. Methods in Enzymol 230: 44-57 (1994).
Uchida, Y., Y. Tsukada and T. Sugimori. Enzymatic properties of neuraminidases from Arthrobacter ureafaciens. J Biochem (Tokyo) 86:573-585 (1979).
Zhang W, Wang H, Zhang L, Yao J, Yang P. Large-scale assignment of N-glycosylation sites using complementary enzymatic deglycosylation. Talanta. Jul 15;85(1):499-505 (2011).