Neuraminidase is the common name for Acetyl-neuraminyl hydrolase (Sialidase). α2-3,6,8 neuraminidase is an exoglycosidase with high purity and stability, which is suitable for the effective release of sialic-acid residues at the non-reducing end on glycoproteins in proteomics and glycobiology. This Neuraminidase catalyzes the hydrolysis of α2-3, α2-6 and α2-8 linked N-acetyl-neuraminic acid residues from glycoproteins and oligosaccharides.
PREPARATION and SPECIFICATION
This enzyme is useful for release of sialic-acid residues at the non-reducing end on glycoproteins in proteomics and glycobiology.
One unit is defined as the amount of enzyme which hydrolyze 1 μmol of sialic-acid per minute under the conditions described below.
Reagent I: 50 mM pH 7.5 potassium phosphate buffer.
Reagent II: 8.0 mM N-Acetylneuraminosyl-D-lactose (Dissolve 52.5mg of 3’-sialylactose in 10 mL of precooled reagent I in ice bath for standby).
Reagent III: 100U/mL NAL: N-acetylneuraminic acid aldolase was diluted with precooled reagent I to 100U/ ml, use ice bath for standby, and store at 4 ℃.
Reagent IV: 100U/ml LDH: Dilute L-LDH (L-lactate dehydrogenase) to 100U/mL with precooled reagent I, use ice bath for standby, and store at 4 ℃.
Reagent V: 2.5 mM NADH: 17.8mg NADH dissolve in 10 mL deionized water and store at -20 ℃ after sub packaging, Thaw before use and use ice bath for standby.
Reagent VI: Enzyme diluent: Dissolve 500 mg BSA in 100 mL reagent I until the final concentration is 0.5 % (W/V). Use ice bath for standby and store at 4 ℃.
Sample: dilute the enzyme with enzyme diluent to 0.1- 0.25 U/mL.
- Pipette Reagent I 125μL, Reagent II 300μL, Reagent V 50μL, Reagent III 62.5μL and Reagent IV 62.5μL fully mix into a cuvette and equilibrate at 37 ℃ for about 2 minutes.
- Add 25 μL of the enzyme solution to the cuvette and mix at 37 ℃ for 10 min.
- Record the ΔAs at 340 nm in 7-10 minute in a spectrophotometer thermostated at 37 ℃.
* At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.
∆A = ∆As – ∆Ab