拟南芥蛋白半衰期的检测

Hongqing  Guo; Yanhai Yin

doi:10.21769/BioProtoc.3318

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Peer-reviewed

Measuring Protein Half-life in Arabidopsis thaliana

拟南芥蛋白半衰期的检测

Hongqing Guo email

Yanhai Yin

发布: 2019年08月05日第9卷第15期 DOI: 10.21769/BioProtoc.3318 浏览次数: 5299

评审: ANU PRIYA MINHASRunlai HangAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Current Biology, featuring study using the protocol.

Oct 2018

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Abstract

Post-translational modifications play important roles in controlling protein function and can lead to altered protein stability. Protein stability can be determined after treatment with the protein synthesis inhibitor Cycloheximide. Cycloheximide is a translational inhibitor that inhibits protein synthesis via cytoplasmic ribosomes. Here we describe how to measure the stability of MYC2 in the context of regulation by FERONIA receptor kinase. First, we describe how to measure MYC2 stability in wild-type and feronia mutant; then we describe similar assays in transgenic plants expressing MYC2-FLAG and MYC2^A12-FLAG (12 FERONIA phosphorylation sites are mutated to Alanine and the mutant protein is stabilized). MYC2 can be induced by mechanical touch, which can be a confounding factor in protein level measurement. In this protocol, we take that into consideration and try to achieve more accurate measurement.

Keywords: FERONIA (FERONIA)

Receptor Kinase (激酶受体)

MYC2 (MYC2)

Transcription factor (转录因子)

Jasmonic acid signaling (茉莉酸信号)

Plant immunity (植物免疫)

Protein phosphorylation (蛋白磷酸化)

Protein degradation (蛋白质降解)

Protein half-life (蛋白半衰期)

Background

We have shown that FERONIA receptor kinase can phosphorylate and destabilize MYC2, a major transcription factor in Jasmonic Acid (JA) signaling, which positively contributes to plant immunity (Guo et al., 2018). Under normal growth conditions, MYC2 is present at very low levels. Overnight treatment with MG132 allows MYC2 to accumulate to a higher level before Cycloheximide treatment (Jung et al., 2015; Jeong et al., 2017), which proves to be important for detection of such unstable proteins. Additionally, MYC2 can be induced by mechanical touch, which can lead to inconsistency in the protein level measurement. Here we provide a detailed protocol to help determine the half-life of proteins like MYC2. We illustrate how this approach can be used with both the endogenous protein and epitope-tagged proteins in transgenic plants. This protocol provides some general guidelines for determining the levels of proteins that exist at low levels and are sensitive to handling (e.g., mechanical touch).

Materials and Reagents

Note: For a complete list of reagents, please refer to Guo et al. (2018).

Pipette tips (MidSci, catalog number: AVR96 for 200 μl tips, AVR96-12 for 1.2 ml tips)
Micropore tape (3M, catalog number: 1530-0)
Kimwipes (Fisher Scientific, catalog number: 06-666A)
Plastic blue pestle (Fisher Scientific, catalog number: 12-141-368)
24-well culture plate (Fisher Scientific, catalog number: 07-200-686)
Nitrocellulose membrane (Bio-Rad, catalog number: 1620112)
Cycloheximide (Sigma-Aldrich, catalog number: C4859)
MG132 (Sigma-Aldrich, catalog number: M7449)
Rabbit polyclonal anti-MYC2 (Guo et al., 2018)
Rabbit polyclonal anti-FLAG (Sigma-Aldrich, catalog number: F7425; RRID: AB_439687)
Goat anti-rabbit IgG-HRP secondary antibody (Thermo Fisher, catalog number: 31460)
Ponceau S (Sigma-Aldrich, catalog number: P7170-1L)
Liquid nitrogen (Chemstore on campus)
DMSO (Fisher Scientific, catalog number: D159-4)
Linsmaier and Skoog packet (CaissonLabs, catalog number: LSP03-1LT)
Sucrose (Fisher Scientific, catalog number: BP220-10)
Tris base (Fisher Scientific, catalog number: BP152-10)
SDS (Fisher Scientific, catalog number: BP166-5)
Glycerol (Fisher Scientific, catalog number: BP229-1)
Bromophenol blue (Fisher Scientific, catalog number: BP115-25)
2-Mercaptoethanol (Fisher Scientific, catalog number: BP176-100)
½ MS liquid medium (see Recipes)
2x SDS sample buffer (see Recipes)