Neuraminidase (Sialidase) Protocol

Neuraminidase Protocol

Using neuraminidase (Product No. 10269611001) to remove sialic acid from purified lipooligosaccharide (LOS)

The enzyme can be used to cleave sialic acids from proteins. A mixture of N-glycosidase F, O-glycosidase, and neuraminidase is often useful (O-glycans often have sialidized structures that could be hydrolyzed by neuraminidase and then O-glycosidase could continue hydrolyzing the structure). The enzyme/substrate ratio should be approx. 0.04 U/25 - 80 μg.

Combination with O-Glucosidase

To use neuraminidase from Vibrio cholerae (instead of neuraminidase from Arthrobacter) together with O-glycosidase under the same conditions, consider the pH-optima of O-glycosidase and the V. cholerea neuraminidase, and therefore, use pH 6 instead of pH 7.2.

Treatment of Formalin-fixed Cells with Neuraminidase

The following procedure adapted from Corral, et al., (1990) BBRC 172:1349-1356:

• Wash 3x10⁷ cells in Dulbecco′s PBS
• Fix cells for 1 hour at +4 °C in an equal amount of 1% paraformaldehyde, 0.1 M sodium-cacodylate, pH 7.3
• Wash cells three times with PBS and resuspend them in PBS at a concentration of 3x10⁷ cells /mL
• Use 150 μL of this cell suspension and treat for 1 hour at +37 °C with 250 U/mL neuraminidase from Vibrio cholerea

Specific inhibitors of neuramindase (silaidase) from Vibrio cholerae:

• 2,3-Dehydro-2-deoxy-N-acetylneuraminic acid
• Higher homologues of the dehydro-deoxy-N-acyl-neuraminic acid series (acyl = propionyl...)
• Di-isopropyl-fluoro-phosphate

It has not been determined whether SDS, guanidine HCl, or urea inhibit the enzyme, but inactivation is possible.

It has not been determined whether CHAPS, Triton X100/X114, 2-mercaptoethanol, or Manidet P40 inhibit the enzyme, but most likely no inhibition occurs. No information is available on inhibition by DTT.