HomeProtein Labeling & ModificationNeuraminidase (Sialidase) Protocol

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.

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