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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.

Materials
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