Mouse NADPH-oxidase 
FOR RESEARCH USE ONLY 
Assay range：0.8U/L – 24U/L 
96 determinations
Purpose
This  kit  allows  for  the  determination  of  NADPH-oxidase  concentrations  in  Mouse 
serum, cell  culture supernates and other biological fluids
Principle of the assay
The  kit   assay  Mouse   NADPH-oxidase  level   in  the   sample,   use  Purified   Mouse 
NADPH-oxidase antibody to coat microtiter plate wells, make solid-phase antibody, then add 
NADPH-oxidase  to   wells,  Combined  NADPH-oxidase   antibody  which  With   HRP  labeled, 
become antibody - antigen - enzyme-antibody complex, after washing Compley, Add TMB 
substrate  solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is 
terminated  by  the  addition  of  a  sulphuric  acid  solution  and  the  color  change  is  measured 
spectrophotometrically    at   a    wavelength   of    450    nm.   The    concentration   of    Mouse 
NADPH-oxidase in the samples is then determined by comparing the O.D. of the samples to 
the standard curve. 
Materials provided with the ki

1.   extract   as  soon  as  possible   after  Specimen  collection,and  according  to   the  relevant 
literature, and  should be experiment as soon as possible after the extraction.       If it can’t, 
specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles. 
2.   Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active. 
Assay procedure
1.   Dilute and add sample:Dilute Original density Standard as follow table: 
24U/L 
2.  Add  sample:  Set  blank  wells  separay  (blank  comparison  wells  don’t  add  sample  and 
HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample 
dilution  40μl  to  testing  sample  well,  then  add  testing  sample  10μl  (sample  final  dilution  is 
5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix. 
3. Incubate:    After closing plate with Closure plate membrane , incubate for 30 min at 37℃. 
4. Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled 
water and reserve. 
5.  Washing:  Uncover  Closure  plate  membrane,  discard  Liquid,  dry  by  swing,  add  washing 
buffer to every well, still for 30s then drain, repeat 5 times, dry by pat. 
6. Add enzyme: Add HRP-Conjugate reagent 50μl to each well, except    blank well. 
7. Incubate: Operation with 3. 
8. Washing: Operation with 5. 
9. Color: Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the 
light preservation for 10 min at 37℃
10.  Stop  the  reaction:  Add  Stop  Solution50μl  to  each  well,  Stop  the  reaction(the  blue  color 
change to yellow color). 
11. Assay: take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and 
within 15min.Steps description

Standard, Sample diluent
Add Standard, Sample diluent, incubate for 30 min at 37℃.
Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃.
Wash 5 times,Add Chromogen Solution A and B, incubate for 10 min at 37℃.
Add Stopp Solution
Read absorbance at 450nm within 15 min
calculate
Calculate
Take the standard density as the horizontal,     the OD value for the vertical ,draw the 
standard curve on graph paper, Find out the corresponding density according to the sample 
OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line 
regression equation of the standard curve with the standard density and the OD value ,with the