Verification of In ...

实验概要

Environmental stresses often trigger rapid generation of reactive oxygen species (ROS) in plants. Excessive amount of ROS can cause damage to plant cells and thus need to be counteracted by cellular antioxidant systems. On the other hand, ROS also serves signaling molecules that modulate various physiological responses and developmental processes. Signaling function of ROS is largely achieved through oxidative modifications of redox-sensitive proteins. Therefore, development of methods for high-throughput identification of redox-sensitive proteins and for verifying and characterizing their in vivo redox states is essential for advancing our understanding of ROS-mediated signaling pathways.

主要试剂

1. Liquid 1/2 MS medium: 1/2 MS salts, 5 g/L sucrose, adjusted to pH 5.7 by KOH.

2. Urea buffer: 8 M urea, 100 mM Tris–HCl (pH 8.2), 1 mM EDTA.

3. 600 mM IAA stock solution in 1 M Tris–HCl, pH 8.2, prepared just before use.

4. 200 mM IAM stock solution in 1 M Tris–HCl, pH 8.2, prepared just before use.

5. Perfect-FOCUS™ (G-Biosciences).

6. Running gel: 8 M urea, 9% (w/v) acrylamide, 0.27% (w/v) bisacrylamide, and 0.037 M Tris–HCl (pH 8.8).

7. Stacking gel: 8 M urea, 2.5% (w/v) acrylamide, 0.075% (w/v) bisacrylamide, and 0.12 M Tris–HCl (pH 6.8).

8. 2× Sample buffer: Urea buffer supplemented with 3.5 mM DTT, 16% glycerol, and 0.2% bromphenol blue.

9. Monoclonal ANTI-FLAG^® M2-Peroxidase (HRP) antibody (Sigma).

实验步骤

1. Expression of Your Favorite Gene-FLAG Fusion Construct in Arabidopsis

   1) Clone Your Favorite Gene (YFG) coding sequence into an appropriate binary vector in which YFG CDS is fused inframe with a FLAG tag, driven by the 35S promoter (see Notes 1 and 2). The resulting construct is referred to as p35S::rFG-FLAG hereafter.

   2) Transform the Agrobacterium strain C58C1 with the 35S:: YFG-FLAG construct.

   3) Transform wild-type Arabidopsis plants by the floral dip method with C58C1 containing 35S: YFG-FLAG and collect T1 seeds 4 weeks after transformation.

   4) Screen T1 plants with appropriate antibiotics according to the selection marker used. Obtain at least ten independent transgenic T1 lines.

   5) Find one transgenic T1 plant with detectable expression level of YFP-FLAG fusion protein by Western blot analysis using the monoclonal ANTI-FLAG^®M2-Peroxidase (HRP) antibody (Sigma).

2. Plant Growth and Treatment

   1) Put about 20 surface-sterilized homozygous transgenic seeds in 50 ml of liquid 1/2 MS medium in each 200-ml flask. After stratification at 4℃ for 3 days, seedlings are grown with gentle (50 rpm) shaking at 22℃ under short-day conditions (9/15-h photoperiod with a light intensity of 100 mol/m2/s).

   2) For theH₂O₂ treatment, a 0.5MH₂O₂ stock solution is added to the flask containing 2-week-old seedlings to a final concentration of 5 mM. For control samples, the same amount of H₂O is added to the flask. Ten minutes after the H₂O₂ addition, plants are harvested and immediately frozen in liquid nitrogen for further analysis (see Note 3).

3. Labeling of YFG-FLAG Fusion Protein with IAA and IAM

   1) 200 mg of plant tissue is homogenized in 1 ml of urea buffer [8 M urea, 100 mM Tris (pH 8.2, 1 mM EDTA) containing 30 mM IAA].

   2) Incubate the sample at 37℃ for 10 min, and then centrifuge at 15,000 × g for 10 min.

   3) Purify protein samples using Perfect-FOCUS™ in order to remove residual IAA.

   4) Resuspend the final acetone precipitate (you did not mention about acetone precipitation earlier) in 95 ml of urea buffer containing 3.5 mM DTT. And then incubate samples at 37℃ for 30 min.

   5) Add IAM to a final concentration of 10 mM. Incubate at 37℃ for 15 min.

   6) For migration markers, 200 mg of seedlings are homogenized in 1 ml of urea buffer containing 3.5 mM DTT. incubation at 37℃ for 30 min, the samples are divided into two Eppendorf tubes and in each tube, either 30 mM IAA or 10 mM IAM is added. Samples are incubated at 37℃ for 15 min.

4. Urea-PAGE and Western Blotting

   1) Cast the running gel andstackinggel as described inSubheading2.

   2) Protein samples were mixed with equal volumes of 2× sample buffer and were loaded into wells.

   3) The gel was run at a constant current of 5mMfor an appropriate period of time in the chamber buffer described in Subheading 2.

   4) Transfer protein from gel to PVDF or nitrocellulose membrane.

   5) Immunoblot detection of YFG-FLAG fusion protein with monoclonal ANTI-FLAG^® M2-Peroxidase (HRP) antibody according to the manufacturer’s instructions.

注意事项

1. Here, we use Flag tag as an example, as the antibody to this epitope tag is commercially available and highly specific. Other epitope tags may be used.

2. Native promoters can also be used if the fusion protein can be readily detected byWestern blotting when expressed under the control of the native promoters. Otherwise, a strong promoter, such as the 35S promoter, is preferred.

3. This protocol can be adapted for identification and characterization of oxidatively modified proteins following other types of treatments leading to oxidative modification of proteins, such as salicylic acid, flagellin, plant pathogens, ABA, and abiotic stresses.