参见作者原研究论文

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May 2016

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Detection of Catalase Activity by Polyacrylamide Gel Electrophoresis (PAGE) in Cell Extracts from Pseudomonas aeruginosa
聚丙烯酰胺凝胶电泳(PAGE)检测铜绿假单胞菌细胞提取物中的过氧化氢酶活性   

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Abstract

Bacteria in nature and as pathogens commonly face oxidative stress which causes damage to proteins, lipids and DNA. This damage is produced by the action of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), singlet oxygen, superoxide anion and hydroxyl radical. ROS are generated by antimicrobials, environmental factors (e.g., ultraviolet radiation, osmotic stress), aerobic respiration, and host phagocytes during infective processes. Pseudomonas aeruginosa, a versatile bacterium, is a prevalent opportunistic human pathogen which possesses several defense strategies against ROS. Among them, two catalases (KatA and KatB) have been well characterized by their role on the defense against multiple types of stress. In this protocol, KatA and KatB activities are detected by polyacrylamide gel electrophoresis (PAGE). It is also suggested that the detection of KatB is elusive.

Keywords: Pseudomonas aeruginosa (铜绿假单胞菌), Catalase (过氧化氢酶), PAGE (PAGE), KatA (KatA), KatB (KatB), H2O2 ( H2O2), Oxidative stress (氧化应激), ROS (ROS)

Background

P. aeruginosa is a ubiquitous bacterium that can be found in a free form in terrestrial and aquatics habitats or as an opportunistic human pathogen causing fatal infections in immunocompromised individuals, patients with skin damage or cystic fibrosis. To defend itself from ROS generated by its strong aerobic metabolism, host phagosomal vacuoles and environmental factors, this microorganism possesses several antioxidative strategies. Among them, two monofunctional catalases (KatA and KatB) are responsible for decomposing H2O2 to water and O2. KatA is the main catalase and has unique characteristics: it is unusually stable and essential to H2O2 resistance, osmoprotection and virulence (Hassett et al., 2000; Lee et al., 2005). It has been suggested that the stability of KatA is one of the main factors for the high level activity under normal growth conditions, and for this reason, katA has been regarded as a constitutively expressed gene in P. aeruginosa (Heo et al., 2010). However, it has been reported that KatA activity is induced in the stationary growth phase (up to 10-fold) and by increased levels of H2O2 (Brown et al., 1995; Suh et al., 1999; Heo et al., 2010). Moreover, katA expression has been demonstrated to be modulated by the global regulator OxyR and Quorum Sensing, whose activation depends on increased levels of H2O2 and high cell density, respectively (Hassett et al., 1999; Heo et al., 2010). KatB is only detected in the presence of H2O2 or paraquat and is partially involved in resistance to oxidative stress (Brown et al., 1995; Lee et al., 2005).

Solar ultraviolet-A (UVA) radiation is one of the main environmental stress factors for P. aeruginosa. Given the oxidative nature of UVA damage, we studied the role of catalases in defense of this microorganism against radiation. We demonstrated that KatA is essential in the optimal response against lethal doses of UVA, both in planktonic cells and biofilms (Costa et al., 2010; Pezzoni et al., 2014). In addition, we reported that low doses of UVA increase KatA and KatB activity and that this regulation occurs at the transcriptional level (Pezzoni et al., 2016). This phenomenon is relevant since it constitutes an adaptive mechanism that prevents cell damage by subsequent exposure to lethal doses of UVA, H2O2, or sodium hypochlorite (Pezzoni et al., 2016).

In the course of our studies, it became necessary to do an in-depth analysis of catalase activity. The total catalase activity in cell extracts was quantified by following spectrophotometrically the decomposition of H2O2, according to Aebi (1984). However, this assay cannot distinguish between KatA and KatB activities. To analyze individual catalase activity, we implement the method proposed by Wayne and Díaz (1986). In brief, crude cell extracts are loaded onto non-denaturing polyacrylamide gels (PAGE), and both catalases are separated by their differential electrophoretic motility; colorless bands of catalase activity are revealed by incubation of the gel with H2O2 and subsequent addition of a ferric chloride-potassium ferricyanide solution. The principle of this method involves the reaction of H2O2 with potassium ferricyanide (III) by reducing it to ferrocyanide (II); the peroxide is oxidized to O2. Ferric chloride reacts with ferrocyanide (II) to form an insoluble blue pigment. Because of the action of catalase on H2O2 decomposition, areas where this enzyme is active develop as clear bands in a blue gel (Patnaik et al., 2013). Additional papers were consulted to fine-tune this technique (Brown et al., 1995; Hassett et al., 1999; Elkins et al., 1999). The studies were performed with the prototypical P. aeruginosa strain PAO1 and isogenic derivatives PW8190 (katA::IslacZ/hah) and PW8769 (katB::IslacZ/hah) carrying mutations into katA and katB, respectively. Mutant strains devoid for KatA or KatB are useful to analyze the role of each enzyme in response to stress and as controls in PAGE catalase assays.

In this protocol, we describe how to detect individual catalase activity by PAGE using cell extracts from P. aeruginosa. Because of the particular characteristics of KatA (high abundance and stability), its detection does not present major difficulties. On the contrary, KatB detection is elusive, so that two changes were applied to the conventional technique: a protein extraction reagent was used instead of sonication to prepare the cell extracts, and the electrophoresis was performed at 4 °C. Based on these assays, it was concluded that KatB is an unstable enzyme, a fact that should be taken into account in quantitative or qualitative catalase assays under inducing (oxidative) conditions.

Materials and Reagents

  1. Pipette tips
  2. 50 ml sterile conical Falcon tubes (Nunc® EZ FlipTM, Thermo Fisher Scientific, catalog number: 362696 )
  3. 1.5 ml sterile Eppendorf centrifuge tubes (Eppendorf, catalog number: 022364111 )
  4. Sonication device
    Note: This was assembled in our laboratory by attaching four plastic tubes (3 cm diameter, 3 cm high) to a plastic box (Figure 1).


    Figure 1. Sonication device

  5. Paper towel (WypAll* X 60 Jumbo Roll, KCWW, Kimberly-Clark, catalog number: 30218593 )
  6. Spatula
  7. Pyrex tray (Pyrex® Storage 13 x 18 cm)
  8. P. aeruginosa strains
    Note: PAO1, referred to as the wild-type, and catalase mutants PW8190 and PW8769 were obtained from the Washington Genome Center. Catalase mutants were constructed by insertion of IslacZ/hah transposon into katA (PW8190, hereinafter KatA-less strain) or katB (PW8769, hereinafter KatB-less strain) (Jacobs et al., 2003).
  9. Distilled water
  10. Tryptone (Oxoid, catalog number: LP0042 )
  11. Yeast extract (Merck, catalog number: 103753 )
  12. Sodium chloride (NaCl) (Biopack, catalog number: 1646.08 )
  13. Albumin from bovine serum (Sigma-Aldrich, catalog number: A4378 )
  14. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
  15. Sodium phosphate (NaH2PO4) (Sigma-Aldrich, catalog number: S0751 )
  16. Hydrogen peroxide (H2O2) 30% (Merck, catalog number: 107210 )
  17. Bugbuster Protein Extraction Reagent (Merck, Novagen, catalog number: 70584-4 )
  18. Sodium thiosulfate (Na2S2O3) (Avantor Performance Materials, MacronTM, catalog number: 8100-04 )
  19. Ammonium persulfate ((NH4)2S2O8) (MP Biomedicals, catalog number: 04802811 )
  20. TEMED (MP Biomedicals, catalog number: 02195516 )
  21. Trizma base (Tris[hydroxymethyl]aminomethane) (C4H11NO3) (Sigma-Aldrich, catalog number: T1503 )
  22. Glycine (Sigma-Aldrich, catalog number: G7126 )
  23. Hydrochloric acid fuming 37% (HCl) (Merck, catalog number: 100317 )
  24. Acrylamide (Sigma-Aldrich, catalog number: A8887 )
  25. Bisacrylamide N,N’-methylene-bis-acrylamide (Sigma-Aldrich, catalog number: M7256 )
  26. EDTA (Merck, Calbiochem, catalog number: 324503 )
  27. Sodium hydroxide (NaOH) (Avantor Performance Materials, MacronTM, catalog number: 7708 )
  28. Glycerol (Merck, catalog number: 104094 )
  29. Bromophenol blue (VWR, DBH, catalog number: 20015 )
  30. Ferric chloride (FeCl3) (Avantor Performance Materials, MacronTM, catalog number: 5029-04 )
  31. Potassium ferricyanide (K3Fe (CN)6) (UCB, catalog number: b1599 )
  32. LB medium (see Recipes)
  33. 4 M NaCl (see Recipes)
  34. Saline solution (see Recipes)
  35. 50 mM sodium phosphate buffer, pH 7 (see Recipes)
  36. 30 mM H2O2 (see Recipes)
  37. 4 mM H2O2 (see Recipes)
  38. 10% ammonium persulfate (see Recipes)
  39. 1.5 M Tris-HCl buffer pH 8.8 (see Recipes)
  40. 1.5 M Tris-HCl buffer pH 6.8 (see Recipes)
  41. 30% acrylamide mix solution (acrylamide bisacrylamide ratio 37.5:1) (see Recipes)
  42. 6% resolving gel solution (see Recipes)
  43. 5% stacking gel solution (see Recipes)
  44. 1 M Tris-HCl buffer pH 8 (see Recipes)
  45. 0.5 M EDTA pH 8 (see Recipes)
  46. Loading sample buffer (see Recipes)
  47. Running buffer (see Recipes)
  48. Ferric chloride/potassium ferricyanide solution (see Recipes)

Equipment

  1. 50, 125 and 150 ml sterile Erlenmeyer flasks (DWK Life Sciences, Duran®, catalog numbers: 21 216 17 , 21 216 28 , 21 990 27 )
  2. 2-20 µl, 20-100 µl, 100-1,000 µl Kartell pluripet micropipettes (Kartell LABWARE, catalog numbers: 13000 , 13210 , 13220 ) and 1-10 ml Acura® manual micropipette (Socorex, model: Acura® manual 825 / Acura® manual 835 )
  3. 50, 100 and 1,000 ml borosilicate measuring cylinders (VILABO, catalog number: 3501114 , 3501115 , 3501118 )
  4. Conventional incubator shaker (New Brunswick Scientific, model: G25 )
  5. Gyratory water bath shaker (New Brunswick Scientific, model: G76 )
  6. Ice maker (Brema, model: TB 551 )
  7. UV-Vis Spectrophotometer (Biotraza, model: 752 )
  8. Refrigerated centrifuge (Hanil Scientific, model: Combi 514R )
  9. Vibra-Cell sonicator (Sonics & Materials, model: VC500 )
  10. Electrophoresis cell (Bioamerica, model: DYCZ-24DNBA )
  11. Power supply (Bioamerica, model: DYY-6CBA )
  12. Freezer ultra-low temperature (Sanyo, model: MDF-U76VC )
  13. Autoclave (HIRAYAMA, HICLAVETM, model: HVE-50 )
  14. Hot air oven sterilizer (Dalvo Intrumentos, model: OHR/T )

Procedure

  1. Preparation of cell extracts
    Non-inducing conditions
    1. Grow Pseudomonas aeruginosa strains (PAO1, KatA-less and KatB-less) overnight in 30 ml of LB medium in 150 ml Erlenmeyer flasks at 37 °C with shaking (200 rpm).
    2. Centrifuge the cultures (20 ml) in 50 ml Falcon tubes for 10 min, 10,000 x g, 4 °C. Discard the supernatants.
    3. Resuspend the cells with 20 ml of cold saline solution and keep them on ice.
    4. Centrifuge for 10 min, 10,000 x g, 4 °C. Discard the supernatants.
    5. Resuspend the cells in ice-cold 50 mM sodium phosphate buffer, pH 7 up to OD650 1 (about 7 ml).
    6. Sonicate 5 ml of concentrated cells in plastic tubes in an ice-water bath (Figure 1) under the following conditions: 2 min in pulsed mode, 18 mm tip diameter, 50% duty cycle, microtip limit 2. Keep the samples on ice.
    7. Centrifuge the extracts for 10 min, 10,000 x g, 4 °C. Discard the pellets carrying unlysed cells and cellular debris. Keep the supernatants on ice for a few hours until using them or store at -80 °C.
    8. Determine protein concentration of the extracts according to Lowry et al. (1951); bovine serum albumin is used as a standard.

    Inducing conditions
    1. Grow Pseudomonas aeruginosa strains (PAO1 and KatB-less) overnight in 30 ml of LB medium in 150 ml Erlenmeyer flasks at 37 °C with shaking (200 rpm).
    2. Dilute overnight cultures in 30 ml of fresh LB medium in 125 ml Erlenmeyer flasks to OD650 0.01 and grow in a gyratory water bath shaker at 37 °C until the cultures reach an OD650 0.3. This OD is reached in about 2 h.
    3. Divide the cultures into two fractions of 10 ml each in 50 ml Erlenmeyer flasks. Maintain one of them untreated (control) and add 2.26 µl of 30 mM H2O2 to the other fraction every 10 min for 1 h under sterile conditions, while shaking both fractions at 37 °C.
    4. Take 5 ml of the treated cultures at the end of 1 h and add 10 μl of 1 mg/ml sodium thiosulfate to neutralize the effect of H2O2; it is not necessary to keep the samples on ice.
    5. Centrifuge the neutralized cultures and 5 ml of the control cultures for 10 min, 10,000 x g, 4 °C. Discard the supernatants.
    6. Resuspend the cells with 10 ml of ice-cold 50 mM sodium phosphate buffer, pH 7.
    7. Centrifuge for 10 min, 10,000 x g, 4 °C. Discard the supernatants.
    8. Resuspend the cells in 1 ml of BugBuster Protein Extraction Reagent and keep for 15 min at room temperature.
    9. Centrifuge for 10 min, 16,000 x g, 4 °C. Keep the supernatants on ice for a few hours until using them or store at -80 °C.
    10. Determine protein concentration of the extracts according to Lowry et al. (1951); bovine serum albumin is used as a standard. 
    The protocol for inducing conditions is schematized in Figure 2.
    Notes:
    1. Antibiotics are not added to culture media.
    2. BugBuster Protein Extraction Reagent was employed instead of sonication to detect KatB activity. This reagent is capable of cell wall perforation without denaturing soluble proteins. It provides an alternative to mechanical methods such as French Press or sonication for releasing proteins.


    Figure 2. Schematic diagram of the experimental procedure

  2. PAGE
    1. Set the two glass plates with the special wedge frames in the gel casting stand (see Figure 3).


      Figure 3. Electrophoresis cell (Bioamerica)

    2. Pipet the 6% resolving gel solution into the gap between the glass plates.
    3. Wait for 15-30 min until it solidifies.
    4. Pipet the 5% stacking gel solution until overflow.
    5. Insert the comb without trapping air under the teeth. Wait for 15-30 min until it solidifies.
    6. Take the glass plates out of the gel casting stand and set them into the electrophoresis cell.
    7. Pour running buffer into the electrophoresis cell until the buffer level is higher than the top of the (shorter) inner gel plate (i.e., until the buffer covers the wells).
    8. Mix 50 µl of the cell extracts with 2 µl of loading buffer in 1.5 ml Eppendorf tubes and heat them in boiling water for 10 min.
    9. Load 10 µg of protein per sample into each well. Cover the cell with the lid and connect the electrodes to the power supply.
    10. Run the electrophoresis at 15 mA at room temperature (non-inducing conditions, KatA activity) or at 4 °C (inducing conditions, KatB activity).
      Note: Electrophoresis is run at 4 °C for detecting KatB activity. This can be done in a cold room or in a refrigerator. However, this is not necessary for the detection of KatA activity.
    11. Stop PAGE running when the dye front almost reaches the foot line of the glass plate. The run generally takes 4 h.

  3. Gel development
    1. Remove the glass plates from the electrophoresis tank and place them on a paper towel. Separate the plates by using a spatula.
    2. Soak the gel in distilled water in a Pyrex tray for 5 min at room temperature. Discard the water.
    3. Incubate the gel with 100 ml of a solution containing 4 mM H2O2 for 10 min at room temperature.
    4. Remove the solution and wash the gel with 100 ml of distilled water at room temperature.
    5. Soak the gel in 100 ml of a solution containing 1% (w/v) ferric chloride and 1% (w/v) potassium ferricyanide at room temperature.
    6. As soon as the gel turns dark green, remove the ferric chloride/potassium ferricyanide solution and rinse with distilled water to prevent overdevelopment.
    7. Once the dye has been removed, photograph the gel immediately. Storage in distilled water for a few days at 4 °C is possible. Areas of catalase activity show up as clear bands.
    Note: It is not necessary to use a shaker or a nutator for the washing and staining steps.

Data analysis

According to this procedure described above, Figure 4 shows representative images of non-denaturing polyacrylamide gels stained for catalase activity employing non-inducing conditions (A) or inducing conditions with H2O2 (B).


Figure 4. Catalase native PAGE analysis of P. aeruginosa wild type (PAO1) and its derivatives KatA-less and KatB-less strains. A. Extracts of PAO1 (wild-type), KatA-less and KatB-less strains grown under non-inducing conditions. The only activity detected corresponds to KatA since KatB is not expressed under this condition. B. Cultures of PAO1 and KatB-less strains, untreated (-) or treated with sublethal concentrations of H2O2 (+), were analyzed for catalase activity to demonstrate detection of KatB activity in non-denaturing polyacrylamide gels.
Note: The images were originally reported in Pezzoni et al., (2014). Protective role of extracellular catalase (KatA) against UVA radiation in Pseudomonas aeruginosa biofilms. J Photochem Photobiol B: Biol 131, 53-64.

Recipes

  1. LB medium
    Dissolve:
    10 g tryptone
    5 g yeast extract
    5 g NaCl
    Bring the volume up to 1,000 ml in distilled water
    Autoclave at 1 atm for 20 min
  2. 4 M NaCl
    Dissolve 46.7 g NaCl in 200 ml of distilled water
    Autoclave at 1 atm for 20 min
  3. Saline solution
    Mix 7.5 ml sterile 4 M NaCl with 300 ml of sterile distilled water
  4. 50 mM sodium phosphate buffer, pH 7
    Solution A: dissolve 70.99 g Na2HPO4 in 500 ml of distilled water
    Solution B: dissolve 59.98 g NaH2PO4 in 500 ml of distilled water
    Mix 10.6 ml of solution A with 14.4 ml of solution B and add 475 ml distilled water
  5. 30 mM H2O2
    Add 0.340 ml of 30% H2O2 to 100 ml 50 mM sodium phosphate buffer, pH 7
    Prepare fresh for every activity assay
    The solution can be kept at room temperature during the experiment
  6. 4 mM H2O2
    Add 0.045 ml of 30% H2O2 to 100 ml distilled water
    Prepare fresh for every activity assay
  7. 10% ammonium persulfate
    Dissolve 1 g ammonium persulfate in 10 ml distilled water
    Store at -20 °C (shelf life 3 months)
  8. 1.5 M Tris-HCl buffer pH 8.8
    Dissolve 18.5 g Trizma base in 80 ml distilled water
    Adjust to pH 8.8 with concentrated HCl and make up the volume to 100 ml
  9. 1 M Tris-HCl buffer pH 6.8
    Dissolve 12.114 g Trizma base in 80 ml distilled water
    Adjust to pH 6.8 with concentrated HCl and make up the volume to 100 ml
  10. 30% acrylamide mix solution
    Dissolve 60 g acrylamide and 1.6 g bis acrylamide in 200 ml distilled water (acrylamide bisacrylamide ratio 37.5:1)
Notes:
  1. Avoid directly contacting with polyacrylamide, ferricyanide and gels; they need to be handled with care.
  2. The order of adding solutions in the resolving and stacking gels solutions is important to avoid an early polymerization before pouring them between the glass plates.
  1. 6% resolving gel solution
    5.4 ml distilled water
    2 ml 30% acrylamide mix
    2.5 ml 1.5 M Tris (pH 8.8)
    0.1 ml 10% ammonium persulfate
    0.008 ml TEMED
  2. 5% stacking gel solution
    2.1 ml distilled water
    0.5 ml 30% acrylamide mix
    0.38 ml 1.5 M Tris (pH 6.8)
    0.03 ml 10% ammonium persulfate
    0.003 ml TEMED
  3. 1 M Tris-HCl buffer pH 8
    Dissolve 12.114 g Trizma base in 80 ml distilled water
    Adjust to pH 8 with concentrated HCl and make up the volume to 100 ml
  4. 0.5 M EDTA pH 8
    Dissolve 47 g EDTA in 200 ml distilled water
    Adjust to pH 8 with concentrated NaOH and make up the volume to 250 ml
  5. Loading sample buffer
    1 ml 1 M Tris-HCl (pH 8)
    4 ml 0.5 M EDTA (pH 8)
    4 ml glycerol
    25 mg bromophenol blue
    1 ml distilled water
    Store at -20 °C
  6. Running buffer
    Dissolve 3.03 g Trizma base and 14.4 g glycine in 1,000 ml distilled water
    Store at 4 °C (shelf life 3 months)
  7. Ferric chloride/potassium ferricyanide solution
    Dissolve 1 g ferric chloride and 1 g potassium ferricyanide in 100 ml distilled water
    Prepare fresh for every activity assay

Acknowledgments

The excellent technical assistance of Ms. P. Pereyra Schuth is gratefully acknowledged. This protocol is based on the work by Wayne and Díaz (1986). Financial support for this research was received from the Comisión Nacional de Energía Atómica (Argentina). M.P. is investigator at Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina).

Competing interests

The authors declare no conflict of interest.

References

  1. Aebi, H. (1984). Catalase in vitro. In: Methods in Enzymology. Parker, L. (Ed.). London, Academic Press 121-126.
  2. Brown, S. M., Howell, M. L., Vasil, M. L., Anderson, A. J. and Hassett, D. J. (1995). Cloning and characterization of the katB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase: purification of KatB, cellular localization, and demonstration that it is essential for optimal resistance to hydrogen peroxide. J Bacteriol 177(22): 6536-6544.
  3. Costa, C. S., Pezzoni, M., Fernandez, R. O. and Pizarro, R. A. (2010). Role of the quorum sensing mechanism in the response of Pseudomonas aeruginosa to lethal and sublethal UVA irradiation. Photochem Photobiol 86(6): 1334-1342.
  4. Elkins, J. G., Hassett, D. J., Stewart, P. S., Schweizer, H. P. and McDermott, T. R. (1999). Protective role of catalase in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide. Appl Environ Microbiol 65(10): 4594-4600.
  5. Hassett, D. J., Ma, J. F., Elkins, J. G., McDermott, T. R., Ochsner, U. A., West, S. E., Huang, C. T., Fredericks, J., Burnett, S., Stewart, P. S., McFeters, G., Passador, L. and Iglewski, B. H. (1999). Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide. Mol Microbiol 34(5): 1082-1093.
  6. Hassett, D. J., Alsabbagh, E., Parvatiyar, K., Howell, M. L., Wilmott, R. W. and Ochsner, U. A. (2000). A protease-resistant catalase, KatA, released upon cell lysis during stationary phase is essential for aerobic survival of a Pseudomonas aeruginosa oxyR mutant at low cell densities. J Bacteriol 182(16): 4557-4563.
  7. Heo, Y. J., Chung, I. Y., Cho, W. J., Lee, B. Y., Kim, J. H., Choi, K. H., Lee, J. W., Hassett, D. J. and Cho, Y. H. (2010). The major catalase gene (katA) of Pseudomonas aeruginosa PA14 is under both positive and negative control of the global transactivator OxyR in response to hydrogen peroxide. J Bacteriol 192(2): 381-390.
  8. Jacobs, M. A., Alwood, A., Thaipisuttikul, I., Spencer, D., Haugen, E., Ernst, S., Will, O., Kaul, R., Raymond, C., Levy, R., Chun-Rong, L., Guenthner, D., Bovee, D., Olson, M. V. and Manoil, C. (2003). Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 100(24): 14339-14344.
  9. Lee, J. S., Heo, Y. J., Lee, J. K. and Cho, Y. H. (2005). KatA, the major catalase, is critical for osmoprotection and virulence in Pseudomonas aeruginosa PA14. Infect Immun 73(7): 4399-4403.
  10. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J Biol Chem 193(1): 265-275.
  11. Patnaik, S. C., Sahoo, D. K. and Chainy, G. B. (2013). A comparative study of catalase activities in different vertebrates. WebmedCentral Zoology 4(6):WMC004270.
  12. Pezzoni, M., Pizarro, R. A. and Costa, C. S. (2014). Protective role of extracellular catalase (KatA) against UVA radiation in Pseudomonas aeruginosa biofilms. J Photochem Photobiol B 131: 53-64.
  13. Pezzoni, M., Tribelli, P. M., Pizarro, R. A., Lopez, N. I. and Costa, C. S. (2016). Exposure to low UVA doses increases KatA and KatB catalase activities, and confers cross-protection against subsequent oxidative injuries in Pseudomonas aeruginosa. Microbiology 162(5): 855-864.
  14. Suh, S. J., Silo-Suh, L., Woods, D. E., Hassett, D. J., West, S. E. and Ohman, D. E. (1999). Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa. J Bacteriol 181(13): 3890-3897.
  15. Wayne, L. G. and Díaz, G. A. (1986). A double staining method for differentiating between two classes of mycobacterial catalase in polyacrylamide electrophoresis gels. Anal Biochem 157(1): 89-92.

简介

自然界中的细菌和病原体通常会面临氧化应激,导致蛋白质,脂质和DNA的损伤。 这种损害是由活性氧(ROS)如过氧化氢(H 2 O 2 2),单线态氧,超氧阴离子和羟基自由基的作用产生的。 ROS在感染过程中由抗菌剂,环境因素(例如,紫外线辐射,渗透压力),有氧呼吸和宿主吞噬细胞产生。 铜绿假单胞菌是一种多功能细菌,是一种普遍的机会性人类病原体,其具有针对ROS的几种防御策略。 其中,两种过氧化氢酶(KatA和KatB)在防御多种类型的压力方面的作用得到了很好的表征。 在该协议中,通过聚丙烯酰胺凝胶电泳(PAGE)检测KatA和KatB活性。 还有人认为KatB的检测是难以捉摸的。

【背景】 P上。铜绿假单胞菌是一种无处不在的细菌,它可以以游离形式在陆地和水生栖息地中发现,或作为机会性人类病原体在免疫功能低下的个体,皮肤损伤或囊性纤维化患者中引起致命性感染。为了抵御其有氧代谢产生的ROS,寄主吞噬泡和环境因素,这种微生物具有多种抗氧化策略。其中,两个单功能的过氧化氢酶(KatA和KatB)负责将H 2 O分解成水和O 2。 KatA是主要的过氧化氢酶并具有独特的特征:它对H 2 O 2抗性,渗透保护和毒力非常稳定并且是必不可少的(Hassett等人 2000; Lee等人,2005)。有人认为,KatA的稳定性是正常生长条件下高水平活性的主要因素之一,因此, katA 已被视为中的组成型表达基因。 P. (Heluginosa)(Heo et al。,2010)。然而,据报道KatA活性在静止的生长阶段(高达10倍)和H 2 O 2(Brown 等人,1995; Suh等人,1999; Heo等人,2010)。此外,已经证明katA表达被全局调节剂OxyR和Quorum Sensing调节,其激活取决于H 2 O 2 >和高细胞密度(Hassett等人,1999; Heo等人,2010)。 KatB仅在H 2 O 2或百草枯存在下被检测到,并且部分参与对氧化应激的抗性(Brown等人, 1995; Lee等人,2005)。

太阳紫外线-A(UVA)辐射是 P的主要环境压力因素之一。 鉴于UVA损伤的氧化性质,我们研究了过氧化氢酶在防御这种微生物对抗辐射中的作用。我们证明KatA对于浮游细胞和生物膜中的致死剂量UVA的最佳反应是必不可少的(Costa等人,2010; Pezzoni等人,2014年)。,2014 )。另外,我们报道了低剂量的UVA增加KatA和KatB活性,并且这种调节发生在转录水平(Pezzoni等人,2016)。这种现象是相关的,因为它构成适应性机制,其通过随后暴露于致死剂量的UVA,H 2 O 2或次氯酸钠(Pezzoni)而防止细胞损伤, et al。,2016)。

在我们的研究过程中,有必要对过氧化氢酶活性进行深入分析。根据Aebi(1984),通过分光光度法分析H 2 O 2 2的分解量来定量细胞提取物中的总过氧化氢酶活性。然而,该测定不能区分KatA和KatB活性。为了分析单个过氧化氢酶活性,我们实施了Wayne和Díaz(1986)提出的方法。简而言之,将粗制细胞提取物加载到非变性聚丙烯酰胺凝胶(PAGE)上,并且通过它们的差异电泳运动分离两种过氧化氢酶;通过用H 2 O 2 2孵育凝胶并随后加入氯化铁 - 铁氰化钾溶液来揭示过氧化氢酶活性的无色条带。该方法的原理是将H 2 O 2与铁氰化钾(III)反应,将其还原为亚铁氰化物(II)。过氧化物被氧化成O 2。三氯化铁与亚铁氰化物(II)反应形成不溶性的蓝色颜料。由于过氧化氢酶对H 2 O 2分解的作用,该酶有活性的区域在蓝色凝胶中以清晰条带形式发展(Patnaik等人, / ,2013)。为了更好地调整这项技术,有人会咨询更多的论文(Brown等人,1995; Hassett等人,1999; Elkins等人, ,1999)。这些研究是用原型 P进行的。铜绿假单胞菌菌株PAO1和同基因衍生物PW8190( katA :: Is lacZ / hah)和PW8769( katB :: Is lacZ / hah)分别携带突变到 katA 和 katB 。缺乏KatA或KatB的突变株可用于分析每种酶在应激反应中的作用,并作为PAGE过氧化氢酶测定中的对照。

在这个协议中,我们描述了如何使用来自 P的细胞提取物通过PAGE检测单个过氧化氢酶活性。绿脓杆菌。 由于KatA的特点(高丰度和稳定性),其检测不会造成重大困难。 相反,KatB检测是难以捉摸的,因此对常规技术应用两种改变:使用蛋白质提取试剂代替超声处理来制备细胞提取物,并且电泳在4℃下进行。 基于这些测定,得出的结论是KatB是一种不稳定的酶,这是在诱导(氧化)条件下在定量或定性过氧化氢酶测定中应该考虑的事实。

关键字:铜绿假单胞菌, 过氧化氢酶, PAGE, KatA, KatB, H2O2, 氧化应激, ROS

材料和试剂

  1. 移液器吸头
  2. 50ml无菌圆锥形Falcon管(EZ Flip TM,Thermo Fisher Scientific,目录号:362696)。

  3. 1.5 ml无菌Eppendorf离心管(Eppendorf,目录号:022364111)
  4. 声波处理设备
    注意:这是在我们的实验室中通过将四个塑料管(直径3厘米,高3厘米)连接到塑料盒(图1)来组装的。


    图1.超声处理设备

  5. 纸巾(WypAll * X 60 Jumbo Roll,KCWW,Kimberly-Clark,目录号:30218593)
  6. 刮刀
  7. Pyrex托盘(Pyrex®储存13 x 18厘米)
  8. P上。绿脓杆菌菌株
    注:PAO1,被称为野生型,过氧化氢酶突变体PW8190和PW8769获自华盛顿基因组中心。通过将IslacZ / hah转座子插入katA(PW8190,下文中为无KatA的菌株)或katB(PW8769,下文中为KatB无菌株)(Jacobs等,2003)来构建过氧化氢酶突变体。
  9. 蒸馏水
  10. 胰蛋白胨(Oxoid,目录号:LP0042)
  11. 酵母提取物(Merck,目录号:103753)
  12. 氯化钠(NaCl)(Biopack,目录号:1646.08)
  13. 来自牛血清的白蛋白(Sigma-Aldrich,目录号:A4378)
  14. 磷酸二氢钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S3264)
  15. 磷酸钠(NaH 2 PO 4)(Sigma-Aldrich,目录号:S0751)
  16. 过氧化氢(H 2 O 2)30%(Merck,目录号:107210)
  17. Bugbuster蛋白提取试剂(Merck,Novagen,目录号:70584-4)
  18. 硫代硫酸钠(Na 2 S 2 O 3)(Avantor Performance Materials,Macron TM,目录号:8100 -04)
  19. 过硫酸铵((NH 4)2 S 2 O 8)(MP Biomedicals,目录号:04802811)
  20. TEMED(MP Biomedicals,目录号:02195516)
  21. Trizma碱(Tris [羟甲基]氨基甲烷)(C 4 H 11 NO 3)(Sigma-Aldrich,目录号:T1503) />
  22. 甘氨酸(Sigma-Aldrich,目录号:G7126)
  23. 盐酸发烟37%(HCl)(Merck,目录号:100317)
  24. 丙烯酰胺(Sigma-Aldrich,目录号:A8887)
  25. 双丙烯酰胺N,N'-亚甲基双丙烯酰胺(Sigma-Aldrich,目录号:M7256)
  26. EDTA(Merck,Calbiochem,目录号:324503)
  27. 氢氧化钠(NaOH)(Avantor Performance Materials,Macron TM,目录号:7708)
  28. 甘油(Merck,目录号:104094)
  29. 溴酚蓝(VWR,DBH,目录号:20015)
  30. 氯化铁(FeCl 3)(Avantor Performance Materials,Macron TM,目录号:5029-04)
  31. 铁氰化钾(K 3 Fe(CN)6)(UCB,目录号:b1599)
  32. LB培养基(见食谱)
  33. 4 M NaCl(见食谱)
  34. 盐溶液(见食谱)
  35. 50 mM磷酸钠缓冲液,pH 7(见食谱)
  36. 30 mM H 2 O 2(见食谱)
  37. 4 mM H 2 O 2(见食谱)
  38. 10%过硫酸铵(见食谱)
  39. 1.5M Tris-HCl缓冲液pH8.8(参见食谱)
  40. 1.5 M Tris-HCl缓冲液pH 6.8(见食谱)
  41. 30%丙烯酰胺混合溶液(丙烯酰胺双丙烯酰胺比例37.5:1)(见食谱)
  42. 6%分解凝胶溶液(见食谱)
  43. 5%浓缩凝胶溶液(请参阅食谱)
  44. 1M Tris-HCl缓冲液pH 8(见食谱)
  45. 0.5 M EDTA pH 8(见食谱)
  46. 加载样品缓冲液(见食谱)
  47. 运行缓冲区(请参阅食谱)
  48. 三氯化铁/铁氰化钾溶液(见配方)

设备

  1. 50,125和150ml无菌锥形瓶(DWK Life Sciences,Duran,目录号:21 216 17,21216 28,2199027)。
  2. 2-20μl,20-100μl,100-1,000μlKartell pluripet微量移液管(Kartell LABWARE,目录号:13000,13210,13220)和1-10ml Acura手动微量移液管(Socorex,型号:讴歌手册825 /讴歌手册835)
  3. 50,100和1000毫升硼硅酸盐量筒(VILABO,产品目录号:3501114,3501115,3501118)
  4. 常规培养摇床(New Brunswick Scientific,型号:G25)
  5. 旋转式水浴摇床(New Brunswick Scientific,型号:G76)
  6. 制冰机(Brema,型号:TB 551)
  7. 紫外可见分光光度计(Biotraza,型号:752)
  8. 冷冻离心机(Hanil Scientific,型号:Combi 514R)
  9. Vibra-Cell超声仪(Sonics& Materials,型号:VC500)
  10. 电泳细胞(Bioamerica,型号:DYCZ-24DNBA)
  11. 电源(Bioamerica,型号:DYY-6CBA)
  12. 冷冻超低温(三洋,型号:MDF-U76VC)
  13. 高压灭菌器(HIRAYAMA,HICLAVETM,型号:HVE-50)
  14. 热风烘箱灭菌器(Dalvo Intrumentos,型号:OHR / T)

程序

  1. 细胞提取物的制备
    非诱导条件
    1. 在30ml LB培养基中于150ml锥形瓶中在37℃振荡(200rpm)下培养绿脓假单胞菌菌株(PAO1,KatA-少和KatB-少)过夜。
    2. 将培养物(20ml)在50ml Falcon管中离心10分钟,10000×g,4℃。丢弃上清液。
    3. 用20毫升冷盐水溶液重悬细胞并将其保存在冰上。
    4. 离心10分钟,10000×g,4℃。丢弃上清液。
    5. 将细胞重新悬浮于冰冷的50mM磷酸钠缓冲液(pH7)中直至OD6501(约7ml)。
    6. 在下列条件下,在冰水浴中(图1),在塑料管中超声处理5 ml浓缩细胞:脉冲模式下2 min,尖端直径18 mm,占空比50%,微尖端限制2.将样品保存在冰上。
    7. 将提取物离心10分钟,10000×g,4℃。丢弃携带未分离细胞和细胞碎片的小球。将上清液保存在冰上数小时,直至使用或储存在-80°C。
    8. 根据Lowry等人(1951)确定提取物的蛋白质浓度;使用牛血清白蛋白作为标准。

    诱发条件
    1. 在30ml LB培养基中于150ml锥形瓶中在37℃振荡(200rpm)下培养铜绿假单胞菌菌株(PAO1和KatB-less)过夜。
    2. 在125ml锥形瓶中的30ml新鲜LB培养基中将过夜培养物稀释至OD505.01,并在37℃的旋转式水浴振荡器中生长,直到培养物达到OD650 / sub> 0.3。这个OD在2小时内达到。
    3. 将培养物在50ml锥形瓶中分成两份,每份10ml。保持其中一个未处理(对照),并在无菌条件下每10分钟将2.26μl的30mM H 2 O 2 2加入另一部分1小时,同时摇动两者在37℃下的分数。
    4. 在1小时结束时取5ml处理的培养物并加入10μl1mg / ml硫代硫酸钠以中和H 2 O 2 2的作用;没有必要将样品放在冰上。
    5. 将中和的培养物和5ml对照培养物离心10分钟,10000×g,4℃。丢弃上清液。
    6. 用10毫升冰冷的50 mM磷酸钠缓冲液(pH 7)重悬细胞。
    7. 离心10分钟,10000×g,4℃。丢弃上清液。
    8. 将细胞重悬于1毫升BugBuster蛋白提取试剂中,并在室温下保持15分钟。
    9. 离心10分钟,16,000×g,4℃。将上清液保存在冰上数小时,直至使用或储存在-80°C。
    10. 根据Lowry等人(1951)确定提取物的蛋白质浓度;使用牛血清白蛋白作为标准。 
    图2描述了诱导条件的协议。
    注意:
    1. 抗生素不添加到培养基中。
    2. 使用BugBuster Protein Extraction Reagent代替超声处理来检测KatB活性。该试剂能够穿透细胞壁而不会使可溶性蛋白质变性。它提供了一种机械方法的替代方法,例如French Press或用于释放蛋白质的超声处理。


    图2.实验程序原理图

  2. PAGE
    1. 将两块玻璃板放在凝胶浇铸台上,使用特殊的楔形框架(见图3)。


      图3.电泳细胞(Bioamerica)


    2. 吸取6%分辨凝胶溶液到玻璃板之间的缝隙中。
    3. 等待15-30分钟,直到它凝固。
    4. 吸取5%浓缩胶溶液直至溢出。
    5. 插入梳子,不要将空气留在牙齿下面。等待15-30分钟,直到它凝固。
    6. 将玻璃板从凝胶浇铸架中取出并放入电泳池中。
    7. 将运行缓冲液倒入电泳池中,直到缓冲液水平高于(较短的)内部凝胶板的顶部(即
    8. 将50μl细胞提取物与2μl上样缓冲液在1.5ml Eppendorf管中混合,并在沸水中加热10分钟。
    9. 每个样品加入10μg蛋白质到每个孔中。
      使用盖子覆盖电池并将电极连接到电源。
    10. 在室温(非诱导条件,KatA活性)或4°C(诱导条件,KatB活性)下以15mA运行电泳。
      注意:电泳在4°C运行以检测KatB活性。这可以在冷室或冰箱中完成。但是,这对检测KatA活动不是必需的。
    11. 当染料前沿几乎达到玻璃板的底部时停止PAGE运行。运行一般需要4小时。

  3. 凝胶发展
    1. 从电泳槽中取出玻璃板并放在纸巾上。使用抹刀分开盘子。
    2. 在室温下将凝胶浸入Pyrex托盘中的蒸馏水中5分钟。丢弃水。
    3. 在室温下用100ml含有4mM H 2 O 2的溶液孵育凝胶10分钟。
    4. 去除溶液并在室温下用100ml蒸馏水洗涤凝胶。
    5. 在室温下将凝胶浸泡在含有1%(w / v)氯化铁和1%(w / v)铁氰化钾的100ml溶液中。
    6. 凝胶变成深绿色后,取出三氯化铁/铁氰化钾溶液并用蒸馏水冲洗以防止过度发展。
    7. 一旦染料被去除,立即拍摄凝胶。在4°C下在蒸馏水中储存几天是可能的。过氧化氢酶活性区域显示为清晰的条带。
    注意:在洗涤和染色步骤中不需要使用振荡器或章动器。

数据分析

根据上述的这个程序,图4显示了使用非诱导条件(A)或用H 2 O 2 2诱导条件对过氧化氢酶活性染色的非变性聚丙烯酰胺凝胶的代表性图像。 (B)。


图4. p。的过氧化氢酶非天然PAGE分析。绿脓杆菌野生型(PAO1)及其衍生物KatA-less和KatB-less菌株A.在非诱导下生长的PAO1(野生型),KatA-less和KatB-less菌株的提取物条件。检测到的唯一活动与KatA相对应,因为在此情况下不会表示KatB。 B.分析未处理( - )或用亚致死浓度H 2 O 2(+)处理的PAO1和KatB-少的菌株的培养物的过氧化氢酶活性,以证明检测非变性聚丙烯酰胺凝胶中的KatB活性。
注:图像最初是在Pezzoni等人(2014年)中报道的。细胞外过氧化氢酶(KatA)对铜绿假单胞菌生物膜中UVA辐射的保护作用。 J Photochem Photobiol B:Biol 131,53-64。



食谱

  1. LB媒介
    解散:
    10克胰蛋白胨
    5克酵母提取物
    5克NaCl

    在蒸馏水中容量达1000毫升 在1个大气压下高压灭菌20分钟
  2. 4 M NaCl

    溶解200毫升蒸馏水中的46.7克氯化钠 在1个大气压下高压灭菌20分钟
  3. 盐水溶液

    将7.5 ml无菌4 M NaCl与300 ml无菌蒸馏水混合
  4. 50mM磷酸钠缓冲液,pH 7
    溶液A:将70.99克Na 2 HPO 4溶于500毫升蒸馏水中
    溶液B:在500毫升蒸馏水中溶解59.98克NaH 2 PO 4。
    将10.6ml溶液A与14.4ml溶液B混合并加入475ml蒸馏水。
  5. 30mM H 2 O 2:n / 2 向100ml 50mM磷酸钠缓冲液(pH 7)中加入0.340ml 30%H 2 O 2。 准备新鲜的每个活动分析

    在实验过程中,溶液可以保持在室温
  6. 4mM H 2 O 2:2 向100ml蒸馏水中加入0.045ml 30%H 2 O 2:
    准备新鲜的每个活动分析
  7. 10%过硫酸铵
    将1克过硫酸铵溶于10毫升蒸馏水中
    存放在-20°C(保质期3个月)
  8. 1.5M Tris-HCl缓冲液pH8.8
    将18.5克Trizma碱溶解在80毫升蒸馏水中
    用浓盐酸调节至pH8.8,并将体积补足至100ml
  9. 1M Tris-HCl缓冲液pH 6.8
    将12.114克Trizma碱溶于80毫升蒸馏水中
    用浓盐酸调节pH至6.8,并将体积调至100ml
  10. 30%丙烯酰胺混合溶液
    将60克丙烯酰胺和1.6克双丙烯酰胺溶于200毫升蒸馏水中(丙烯酰胺双丙烯酰胺比例为37.5:1)
注意:
  1. 避免与聚丙烯酰胺,铁氰化物和凝胶直接接触;他们需要小心处理。
  2. 在溶解和堆积凝胶溶液中添加溶液的顺序非常重要,可以避免在将玻璃板倒入玻璃板之前进行早期聚合。
  1. 6%分解凝胶溶液
  2. 5.4毫升蒸馏水
    2毫升30%丙烯酰胺混合
    2.5 ml 1.5 M Tris(pH 8.8)

    0.1毫升10%过硫酸铵 0.008毫升TEMED
  3. 5%浓缩胶溶液
    2.1毫升蒸馏水

    0.5毫升30%丙烯酰胺混合物 0.38 ml 1.5 M Tris(pH 6.8)

    0.03毫升10%过硫酸铵 0.003毫升TEMED
  4. 1M Tris-HCl缓冲液pH 8
    将12.114克Trizma碱溶于80毫升蒸馏水中
    用浓盐酸调节pH至8,并将体积调至100 ml
  5. 0.5 M EDTA pH 8

    溶解在200毫升蒸馏水中的47克EDTA
    用浓NaOH调节至pH 8,并将体积补充至250 ml
  6. 加载样本缓冲区
    1毫升1M Tris-HCl(pH 8)
    4毫升0.5M EDTA(pH8)
    4毫升甘油
    25毫克溴酚蓝
    1毫升蒸馏水
    在-20°C储存
  7. 运行缓冲区

    在3.0毫升蒸馏水中溶解3.03克Trizma碱和14.4克甘氨酸 在4°C储存(保质期3个月)
  8. 三氯化铁/铁氰化钾溶液

    1克三氯化铁和1克铁氰化钾溶于100毫升蒸馏水中 准备新鲜的每个活动分析

致谢

感谢P. Pereyra Schuth女士的出色技术支持。该协议基于Wayne和Díaz(1986)的工作。该研究的经济支持来自阿根廷国家能源委员会(阿根廷)。熔点是Consejo Nacional de InvestigacionesCientíficasyTécnicas(CONICET,阿根廷)的调查员。作者声明没有利益冲突。

参考

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  2. Brown,S.M.,Howell,M.L.,Vasil,M.L.,Anderson,A.J。和Hassett,D.J。(1995)。 编码过氧化氢的绿脓杆菌katB基因的克隆和鉴定 - 诱导性过氧化氢酶:KatB的纯化,细胞定位,并证明它对于最佳抗过氧化氢的抗性是必不可少的。
  3. Costa,C. S.,Pezzoni,M.,Fernandez,R. O.和Pizarro,R. A.(2010)。 法定人数感应机制在<铜绿假单胞菌对致命性应答的作用和亚致死UVA照射。 Photochem Photobiol 86(6):1334-1342。
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  6. Hassett,D.J.,Alsabbagh,E.,Parvatiyar,K.,Howell,M.L.,Wilmott,R.W。和Ochsner,U.A。(2000)。 蛋白酶抗性过氧化氢酶KatA在静止期细胞裂解后释放,对于有氧存活在低细胞密度下的铜绿假单胞菌oxyR突变体。 J Bacteriol 182(16):4557-4563。
  7. Heo,Y.J.,Chung,I.Y.,Cho,W.J.,Lee,B.Y.,Kim,J.H.,Choi,K.H.,Lee,J.W.,Hassett,D.J。和Cho,Y.H。(2010)。 绿脓杆菌PA14的主要过氧化氢酶基因(katA)全球反式激活因子OxyR对过氧化氢反应的正面和负面控制。
  8. Jacobs,MA,Alwood,A.,Thaipisuttikul,I.,Spencer,D.,Haugen,E.,Ernst,S.,Will,O.,Kaul,R.,Raymond,C.,Levy,R.,Chun -Rong,L.,Guenthner,D.,Bovee,D.,Olson,MV和Manoil,C。(2003)。 铜绿假单胞菌综合转座子突变库 。 > Proc Natl Acad Sci USA 100(24):14339-14344。
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引用:Pezzoni, M., Pizarro, R. A. and Costa, C. S. (2018). Detection of Catalase Activity by Polyacrylamide Gel Electrophoresis (PAGE) in Cell Extracts from Pseudomonas aeruginosa. Bio-protocol 8(11): e2869. DOI: 10.21769/BioProtoc.2869.
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