Enzymatic CarboRelease Kit

Kit includes the enzymes, controls, and reagents required to remove all N-linked oligosaccharides and many O-linked sugars from 2 mg of glycoprotein in one 3 hour incubation. Enough reagents are included for twenty deglycosylation reactions. The enzymes are packaged seperately in individual 20 microlitre vials. This gives researchers the flexibility to characterize the glycans attached to their glycoprotein more fully than with our mixture of enzymes. Appropriate use of individual glycosidases allow the determination of sialyation (use of Sialidase only) and the specific presence of N-linked glycans (using PNGase F only) and O-linked glycans (using Sialidase, ß-Galactosidase, O-Glycosidase, and Glucosaminidase).
Includes 20 microlitres each of the following enzymes: PNGase F (Chryseobacterium meningosepticum) O-Glycosidase (Streptococcus pneumoniae) Sialidase (Arthrobacter ureafaciens) ß-Galactosidase (Streptococcus pneumoniae) Glucosaminidase (Streptococcus pneumonia)	Other Supplied Reagents: Reaction buffer Denaturation Solution Triton X Bovine Fetuin (control)
Specificity The Enzymatic CarboRelease Kit will remove all N-linked oligosaccharides and many O-linked oligosaccharides from glycoproteins. N-links (Asparganine-linked) are removed 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 Sialidase and O-Glycosidase. The addition of ß-Galactosidase and Glucosaminidase will assist in the deglycosylation of larger O-link structures. Directions 1. Mix 10 µl of reaction buffer with up to 100 µg of glycoprotein in 30 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 1 µl each of PNGase F, Sialidase, ß-Galactosidase, Glucosaminidase, and O-Glycosidase. 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 to 24 hours.