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Neuraminidase > FAQ |  | Neuraminidase FAQ
See the Protein Tools FAQ also.
Q1: Can I use Neuraminidase in a double digest with Endo H(Hf) or PNGase F?
Q2: Is Neuraminidase active at higher pH levels?
Q3: What is the difference between Neuraminidase and α2-3 Neuraminidase?
Q4: How much exoglycosidase should be used?
Q5: Do detergents inhibit exoglycosidases/endoglycosidases?
Q6: What is a good positive control for neuraminidase?
Q7: Do detergents inhibit exoglycosidases/endoglycosidases?
Q8: What are Glycosidases and their uses?
Q1: Can I use Neuraminidase in a double digest with Endo H(Hf) or PNGase F?
A1: Yes. As with the above question we've observed excellent Neuraminidase activity at higher and lower pH's, and we've also found it to work very well on a protein that has been denatured. Neuraminidase shows normal activity even in the presence of 1% BME, and 0.5% SDS.
Q2: Is Neuraminidase active at higher pH levels?
A2: Yes. Neuraminidase has a very broad pH curve. Although optimum pH is around 6.0, we see excellent sialic acid removal at pH's as low as 4.5 and as high as pH 8.5. We have used Neuraminidase at pH ranges more suitable for PNGase F (pH range 7.5-8.6).
Q3: What is the difference between Neuraminidase and α2-3 Neuraminidase?
A3: α2-3 Neuraminidase has a >500 fold preference for α2-3 linkages over α2-6 and α2-8. Neuraminidase cleaves α2-3, α2-6 and α2-8 linkages.
Q4: How much exoglycosidase should be used?
A4: The amount of enzyme required varies when different substrates are used. Start with 1-2 µl for 1µg of glycoprotein or 100 nM of oligosacharide for one hour in a 10-25 µl reaction. If there is still undigested material, let the reaction go overnight.
Q5: Do detergents inhibit exoglycosidases/endoglycosidases?
A5: At moderate levels (0.5-1.0% ionic and non-ionic detergents) most of the glycosidases show satisfactory activity or are unaffected. One major exception is PNGase F. SDS inhibits it.
Q6: What is a good positive control for neuraminidase?
A6: Fetuin. pNP-N-acetyl-neuraminic acid
Q7: Do detergents inhibit exoglycosidases/endoglycosidases?
A7: At moderate levels (0.5-1.0% ionic and non-ionic detergents) most of the glycosidases show satisfactory activity or are unaffected. One major exception is PNGase F as it is inhibited by SDS.
Q8: What are Glycosidases and their uses?
A8: Glycosidases are used to get information about the carbohydrate groups attached to glycoproteins and glycopeptides. They come in two varieties, endoglycosidases that cleave entire carbohydrate groups from proteins and exoglycosidases that remove monosaccharides from the nonreduced ends of the carbohydrate. A reduced end is one generated by an endoglycosidase. Researchers frequently use an endoglycosidase followed by one or more exoglycosidases and then analyze the products using SDS-PAGE or various types of liquid chromatography.
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