Search

Cellular Extract Preparation for Superoxide Dismutase (SOD) Activity Assay   

Edited by
Tie Liu
Download PDF How to cite Favorites 2 Q&A Share your feedback Cited by

In this protocol

Original research article

A brief version of this protocol appeared in:
New Phytologist
Jan 2013

Abstract

Superoxide dismutase (SOD) acts as a primary defence against reactive oxygen species (ROS) by converting O2- to O2 and H2O2. Members of this enzyme family include CuZnSOD, MnSOD and FeSOD. Most eukaryotes harbor CuZnSOD and MnSOD, and FeSOD is found in plants and prokaryotes. This protocol is to demonstrate how to prepare the cellular extract for the identification and characterization of SODs in planta.

Keywords: SOD, Activity Assay, N

Materials and Reagents

  1. Nitroblue tetrazolium (NBT) (Sigma-Aldrich, catalog number: N6876 )
  2. Riboflavin (Sigma-Aldrich, catalog number: R4500 )
  3. N,N,N’,N’-Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
  4. KCN (Sigma-Aldrich, catalog number: 60178 )
  5. H2O2 (Sigma-Aldrich, catalog number: 349887 )
  6. NBT solution (see Recipes)
  7. Grinding buffer (see Recipes)
  8. Riboflavin solution (see Recipes)
  9. KCN solution (see Recipes)
  10. H2O2 solution (see Recipes)

Equipment

  1. A light box (white light)
  2. Centrifuge (Heraecus, Biofuge fresco)
  3. Protein gel cassette

Procedure

  1. Arabidopsis cellular extract preparation
    1. Arabidopsis seedlings were grown at 23 °C with 16 h of light at 60–100 μmol m-2 s-1. Nine-day-old seedlings were collected and weighted.
    2. Seedlings were homogenized with ice-cold Grinding buffer (tissue weight/buffer volume = 1 mg/3 μl).
      Note that the tissue and extract should be kept at 4 °C during all extraction processes.
    3. Centrifuge at 16,000 x g at 4 °C for 10 min.
    4. The supernatant is the resulting cellular extract, and the amount of protein was quantified by Bradford method (1976).
  2. SOD activity staining
    1. Proteins or cellular extract (15 to 25 μg) was subjected to 10% native-PAGE at 4 °C.
    2. Wash the gel with distilled water for 3 times.
    3. Incubate with NBT solution in dark with shaking for 15 min at room temperature (RT).
    4. Pour off the NBT solution, wash the gel with distilled water for 3 times.
    5. Incubate with Riboflavin solution in dark with shaking for 15 min at RT.
    6. Pour off the Riboflavin solution, wash the gel with distilled water for 3 times.
    7. Gel was illuminated with a white-light box for 10-15 min at RT. During illumination, immerse gel in a thin layer of distilled water to avoid drying the gel.
    8. Under light exposure, the riboflavin is reduced then leading the production of O2-. NBT is reduced by O2- to form formazan, a dark blue color precipitate. The enriched SOD activity scavenges the O2- to prevent the formation of formazan, thus, the white SOD activity bands appear in the blue background.
  3. Identification of different SOD species (Figure 1)
    1. KCN treatment: KCN inhibits the CuZnSOD activity only.
      All procedures are the same as SOD activity staining processes except the addition of KCN to final 8 mM in Riboflavin solution.
    2. H2O2 treatment: H2O2 inhibits both CuZnSOD and FeSOD activities.
      After native-PAGE and prior to NBT staining of SOD activity staining processes, soak the gel with 8 mM H2O2 solution for 30 min with shaking at room temperature. Wash the gel with distilled water for 3 times, and follow the remaining processes of SOD activity staining.


Figure 1. SOD activity verification in Arabidopsis thaliana. KCN is an inhibitor of CuZnSOD activity, whereas H2O2 inhibits both CuZnSOD and FeSOD activities. MnSOD activity is not inhibited by either treatment.

Recipes

  1. Grinding buffer
    150 mM Tris (pH 7.2)
  2. NBT solution
    0.1% NBT dissolved in distilled water.
    Store in 4 °C in dark
  3. Riboflavin solution (freshly prepare before use)
    28 μM riboflavin and 28 mM TEMED in 0.1 M potassium phosphate buffer (pH 7.0).
  4. 2 N KCN solution
    KCN in distilled water. Store in 4 °C.
  5. 8 mM H2O2 solution (freshly prepare before use)
    Add 27 μl H2O2 (35%) into 30 ml 0.1 M potassium phosphate buffer (pH 7.0).

References

  1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  2. Kuo, W. Y., Huang, C. H., Liu, A. C., Cheng, C. P., Li, S. H., Chang, W. C., Weiss, C., Azem, A. and Jinn, T. L. (2013). CHAPERONIN 20 mediates iron superoxide dismutase (FeSOD) activity independent of its co-chaperonin role in Arabidopsis chloroplasts. New Phytol 197(1): 99-110.
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Kuo, W., Huang, C., Shih, C. and Jinn, T. (2013). Cellular Extract Preparation for Superoxide Dismutase (SOD) Activity Assay. Bio-protocol 3(13): e811. DOI: 10.21769/BioProtoc.811.
Q&A

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

gary jefferson
UBS
Is there any way to purchase SOD Gel. I read the article by Dr. Frank Shallenberger in "Second Opinion", January 2015, which described the significant results using SOD Gel. Unfortunately, I can't locate a source for purchasing it anywhere. Many thanks, Gary
1/7/2015 12:14:14 PM Reply
Tsung-Luo Jinn
Department of Life Science and Institute of Plant Biology, National Taiwan University

You only need is the SDS eliminated 12.5% PAGE (without SDS), which is good for in gel SOD activity assay, best,

1/7/2015 11:16:28 PM


Rashmi Mishra
M.S University
DOES SOD GETS DENATURED AT TEMPERATURES HIGHER THAN 4 DEGREE CELCIUS?
1/1/2015 1:16:57 AM Reply
Tsung-Luo Jinn
Department of Life Science and Institute of Plant Biology, National Taiwan University

Hi,
Please extract your sample with ice-cold buffer, and then run your gel in cold room,
Which can give you best result,
Sure, SOD may or may not denature when temp higher then 4C, different SOD from different sample do have it optimal reaction temp and pH value,
Best,

1/4/2015 12:17:37 AM