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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 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 (Asn-linked) are removed using the enzyme PNGase F. In addition, all Ser/Thr-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 Hexosaminidase 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 35 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.
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