Materials and Reagents

Sucrose
HEPES
EDTA
DTT
L-cysteine
MgCl₂
PVP
Complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets (F. Hoffmann-La Roche, catalog number: 04693159001 )
BSA
Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 500-0006 )
Tris-HCl
Sodium 3,3'-( -[(phenylamino)carbonyl] -3,4-tetrazolium)-bis (4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) (Sigma-Aldrich, catalog number: X4626 )
NADPH (Sigma-Aldrich, catalog number: N1630 )
Protein extraction working solution (see Recipes)

Equipment

Microtiter plate reader (Infinite M200 Pro, Tecan)
Microcentrifuge (AqquSpin Micro R) (Thermo Fisher Scientific)
Ultracentrifuge ( Optima TLX , Beckman)
Microtiter plate (BD Biosciences, catalog number: 353075 )

Procedure

Protein extraction and separation of membrane fraction from plant tissues
1. Harvest tissue in liquid nitrogen. If not used immediately, keep at -80 °C until processing.
2. Grind tissue in liquid nitrogen and weigh out 0.5 g of the ground tissues in empty Falcon tube that has been pre-chilled in liquid nitrogen and used to tare the scale.
3. Add 6 ml of ice-cold protein extraction buffer to ground tissues on ice.
4. Vortexat room temperature to mix thoroughly.
5. Filter homogenized tissue through four layers of cheese cloth and transfer filtrate (flow-through) to 2-ml microcentrifuge tubes, on ice.
6. Centrifuge at 10,000 x g for 45 min at 4 °C and transfer supernatant to ultra- centrifuge tube.
7. Separate total membrane fractions by ultra-centrifugation at 203,000 x g for 60 min at 4 °C.
8. Discard supernatant and resuspend pellet in 1 ml ice-cold 10 mM Tris-HCl (pH 7.4).
Protein estimation using Bradford microassay (160 μl)
1. Prepare BSA standards ranging from 5 μg-25 μg/ml as follows:
  
  These standards will be used to generate a standard curve.
2. Use 96 well microtiter plate to prepare reaction mix.
3. Prepare blank byadding 160 μl of water to one well in triplicates.
4. Prepare test samples by adding 2 μl of supernatant (from section 1) to 158 μl of water.
5. Add 40 μl of Bradford Assay reagent to BSA standards, blank and test samples.
6. Mix and incubate at room temperature for 5 min and read absorbance at 595 nm (A₅₉₅) on plate reader spectrophotometer.
  Note: If spectrophotometer does not include a software to generate standard curve to automatically estimate protein content, generate a BSA standard curve by plotting known protein concentration (X-axis) vs. Absorbance (in Y-axis). Protein concentration for a given unknown sample is estimated by plotting the A₅₉₅ absorbance of the unknown (in the y-axis) and determining the intersection point with the BSA standard curve and then find the concentration associated with that particular point (in the x-axis). If using excel, after plotting concentration vs A₅₉₅, obtain the trendline and use the equation for the line and the A₅₉₅ of the unknown to resolve the unknown concentration.
NADPH oxidase activity assay
1. Prepare fresh solution of 1 mM XTT and 1 mM NADPH.
2. Prepare two different assay solutions A and B:
  
  Note: Because membrane fraction can spontaneously reduce XTT, even in the absence of substrate (NADPH), it is necessary to prepare two blanks, one without NADPH, to correct for this background levels of activity.
3. Prepare blanks:
  Blank 1: Add 10 μl of water to 240 μl of Assay solution A.
  Blank 2: Add 10 μl of membrane fraction to 240 μl Assay solution B.
4. Prepare samples by adding 10 μl of membrane fraction (from section 1) to 240 μl of Assay solution A. Read the absorbance at 492 nm (A₄₉₂) at 0 min and then 20 min intervals for one hour or until saturation point reached.
5. To get final A₄₉₂ Blank reading, subtracts A₄₉₂ Blank1 and A₄₉₂ Blank 2.
6. Calculate rate of O^2– generation by using an extinction coefficient 2.16 x 10⁴ cm-(Jiang and Zhang 2002).
  (Δ_A492nm/min _test-Δ_A492nm/min_blank) / (2.16X10^4M-1C^M-1) (0.04)
  Δ_A492nm/min _Test= A_{492 (sample X)} at saturation point - A_{492 (sample X)}at 0 min
  Δ_A492nm/min _blank = A_{492nm (blank)} at saturation point - A_{492nm (blank)} at 0 min
  0.04 = dilution factor (10 μl/250 μl)
  To calculate specific activity, divide the value obtained in equation by the amount of protein present in the sample (converted to mg/ml).

Recipes

Protein extraction working solution
0.25 M sucrose
50 mM HEPES
3 mM EDTA
1 mM DTT
3.6 mM L-cysteine
0.1 mM MgCl₂
0.6% PVP
10 Tablets of Complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets.
Prepare the following stock solutions:
1 M sucrose
1 M HEPES (pH 7.2)
0.25 M EDTA
1 M DTT
100 mM MgCl₂
In 80 ml of water add the following reagents:

Add 10 Tablets of Complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets.
Mix well and adjust volume to 100 ml

Acknowledgments

This protocol has been adapted and modified to use in Arabidopsis from Jiang and Zhang (2002). This work was supported by the Samuel Roberts Noble Foundation.

References

Able, A. J., Guest, D. I. and Sutherland, M. W. (1998). Use of a new tetrazolium-based assay to study the production of superoxide radicals by tobacco cell cultures challenged with avirulent zoospores of phytophthora parasitica var Nicotianae. Plant Physiol 117(2): 491-499.
Jiang, M. and Zhang, J. (2002). Involvement of plasma-membrane NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense in leaves of maize seedlings. Planta 215(6): 1022-1030.
Rojas, C. M., Senthil-Kumar, M., Wang, K., Ryu, C. M., Kaundal, A. and Mysore, K. S. (2012). Glycolate oxidase modulates reactive oxygen species-mediated signal transduction during nonhost resistance in Nicotiana benthamiana and Arabidopsis. Plant Cell 24(1): 336-352.
Sagi, M. and Fluhr, R. (2001). Superoxide production by plant homologues of the gp91(phox) NADPH oxidase. Modulation of activity by calcium and by Tobacco mosaic virus infection. Plant Physiol 126(3): 1281-1290.