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.
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).
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:
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.
|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)|
|Application||Research use only, not for diagnostic use|
Time taken: 30 minutes incubation at 37°C
Sample requirement: Urine/Serum sample
Contains: Rapichrome Substrate V, Incubation Buffer R, Colour Development Buffer A
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)
|50 test kit||1300050|
|100 test kit||1300100|
|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.