NAG Spectrophotometric Method

VRA-NAG Assay Kits

Spectrophotometric measurement of NAG (N-Acetyl-β-D-glucosaminidase) enzyme activity.

Introduction to the VRA-NAG Kit

This kit procedure incorporates the VRA-GlcNAc substrate and provides an alternative to MNP-NAG assay kit when a spectrophotometric measurement is required. This assay gives an intense colour and the end product is more stable.

 

VRA reaction.jpg

Figure 1. VRA-glcNAc substrate for spectrophotometric detection of NAG enzyme

The released colour is stable at 505 nm and this avoids the use of a calibrant. The enzyme reaction is carried out at saturation and this together with the high extinction coefficient (Ɛ= 38,600 Lmol-1cm-1) of the chromophore at 505 nm (pH 9.5) gives this high sensitivity. The procedure has been used for the assay of urine from children where samples are often dilute and high sensitivity is required (see references).

Assay Procedure

Random urine sample (50 µl) is added to substrate solution (700 µl) and incubated for 30 minutes at 37°C. The reaction is stopped by the addition of buffer pH 10. the colour released is measured at 505 nm.

The activity of the enzyme in µmol/min/L of chromophore released is then calculated according to the equation:

VRA equation.jpg

Here, A is the absorbance at 505 nm, Vr (mL) stands for the volume of the reaction mixture after addition of the stopping buffer, 1 (cm) represents the cuvette length, t (min) is the incubation time, and Vs (mL) stands for the applied sample volume.

Specifications

Product code 1300050/1300100
Description NAG assay kit
Species type Human; animal
Sample type Urine; serum; plasma
Number of tests 50/100
Sample volume 50 μL
Sensitivity 10 μmol/h/L; 0.16 U/L
Range 10μmol/h/L (0.16 U/L) to 2000 μmol/h/L (33 U/L)
Incubation 30 mins
Wavelength 505 nm
Application Research use only, not for diagnostic use

Product Description

Time taken: 30 minutes incubation at 37°C

Sample requirement: Urine/Serum sample

Contains: Rapichrome Substrate V, Incubation Buffer R, Colour Development Buffer A

Kit Contents

1. Rapichrome Substrate V: NAG substrate containing Ammonium5-[4-(2-acetamido-2-deoxy-beta-D-glucopyranosyloxy)-3-methoxymethylene]-2-thioxothiazolidin-4-one-3-acetate hydrate (VRA-glcNAc).

2. Incubation Buffer R

3. Colour Development Buffer A

4. Optional – NAG enzyme Controls (Low, Medium and High)

Test for 50 or 100 Samples available (50 ul per sample).

VRA-NAG Assay Kits (Spectrophotometric method)

Description Product Code
50 test kit 1300050
100 test kit 1300100

Click here for MSDS of VRA-GlcNAc substrate

NAG/Creatinine controls*

Description Product Code MSDS
High: NAG 2500-3500 μmol/h/L; creatinine 18 mM 1300008
Medium: NAG 1400-1800 μmol/h/L; creatinine 9 mM 1300009
Low: NAG 750-950 μmol/h/L; creatinine 3 mM 1300010

* These NAG/creatinine controls are for use with VRA-NAG kits only (1300050/1300100). For use with the MNP-NAG kits (1290050/1290100), please click here.