Improve Research Reproducibility A Bio-protocol resource
  • search
  • 提交稿件
  • 订阅
  • CN change language
Peer-reviewed

Using 14C-acetate Pulse-chase Labeling to Study Fatty Acid and Glycerolipid Metabolism in Plant Leaves

用14C -乙酸酯脉冲追逐标记法研究植物叶片中脂肪酸和甘油脂代谢

LY Linhui Yu
CZ Chao Zhou
JF Jilian Fan
CX Changcheng Xu

发布: 2021年02月05日第11卷第3期 DOI: 10.21769/BioProtoc.3900 浏览次数: 3179

评审: Agnieszka ZienkiewiczAnonymous reviewer(s)

download pdf 下载 PDF 下载 PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of The Plant Cell, featuring study using the protocol.

Jul 2019

Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

拟南芥中硝酸盐的荧光生物传感器成像

拟南芥中硝酸盐的荧光生物传感器成像

Yen-Ning Chen and Cheng-Hsun Ho

2023年08月20日 2002 阅读

大麻的无性繁殖及其雌花序中提取得到的树脂

大麻的无性繁殖及其雌花序中提取得到的树脂

Sebastián D´Ippolito [...] Silvana L. Colman

2025年02月20日 725 阅读

植物样本中阴离子磷脂所有分子种类与类别的同步检测及半定量新方法

植物样本中阴离子磷脂所有分子种类与类别的同步检测及半定量新方法

Manon Genva [...] Laetitia Fouillen

2025年04月20日 689 阅读

Abstract

Lipids metabolism is comprised of networks of reactions occurred in different subcellular compartments. Isotopic labeling is a good way to track the transformations and movements of metabolites without perturbing overall cellular metabolism. Fatty acids, the building blocks of membrane lipids and storage triacylglycerols, are synthesized in plastids. The immediate precursor for fatty acid synthesis is acetyl-CoA. Exogenous acetate is rapidly incorporated into fatty acids in leaves and isolated plastids because it can diffuse freely through cellular membranes, enter the plastid where it is rapidly metabolized to acetyl-CoA. Therefore, isotope-labeled acetate is often used as a tracer for the investigation of fatty acid synthesis and complex lipid metabolism in plants and other organisms. The basic principle of isotope labeling and its recent technological advances have been reviewed (Allen et al., 2015). The present protocol describes the use of 14C-labeled acetate to determine rates of fatty acid synthesis and degradation and to track the metabolism of glycerolipids in leaves. This method, which is often referred to as acetate pulse-chase labeling, has been widely used to probe various aspects of lipid metabolism (Allen et al., 2015), including the role of autophagy in membrane lipid turnover (Fan et al., 2019) and the interplay between lipid and starch metabolism pathways (Yu et al., 2018).

Keywords: 14C-acetate pulse-chase (碳14乙酸酯脉冲追踪) search Fatty acid (脂肪酸) search Lipid (脂质) search

Materials and Reagents

  1. Pipette tips

  2. 90 mm Petri dish (Thermo Fisher Scientific, catalog number: 101R20 )

  3. 1.5 ml and 0.5 ml Eppendorf safe-lock microcentrifuge tube

  4. TLC Silica gel 60 plate (Merck)

  5. Filter paper

  6. Pencil

  7. 3-4-week-old Arabidopsis plants

  8. 14C-acetate (American Radiolabeled Chemicals, catalog number: ARC 0101D-1 mCi )

  9. Double-distilled water (ddH2O)

  10. MES (Sigma-Aldrich, catalog number: M3671 )

  11. MS salt (Gentaur, catalog number: LSP03-50LT )

  12. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )

  13. Chloroform (Thermo Fisher Scientific, catalog number: C607 )

  14. Methanol (VWR Chemicals, catalog number: BDH1135-4LG )

  15. Acetic acid (Macron, catalog number: MK-8817-500 )

  16. Acetone (Macron, catalog number: MK244310 )

  17. Toluene (VWR Chemicals, catalog number: BDH1151-4LG )

  18. Hexane (VWR Chemicals, catalog number: BDH1129-4LG )

  19. Diethyl ether (Macron, catalog number: MK085010 )

  20. (NH4)2SO4 (Mallinckrodt Chemicals, catalog number: 3512-12 )

  21. Nitrogen gas

  22. Scintillation liquid (PerkinElmer, catalog number: 6013199 )

  23. Tween-20 (Sigma-Aldrich, CAS number: 9005-64-5 )

  24. H3PO4 (Sigma-Aldrich, CAS number: 7664-38-2 )

  25. Labeling medium (see Recipes)

  26. Lipid extraction solution (see Recipes)

  27. KCl solution (see Recipes)

  28. Developing solvent 1 (see Recipes)

  29. Developing solvent 2 (see Recipes)

Equipment

  1. Pipettes

  2. Analytical balance (Mettler Toledo, model: AE100 )

  3. Shaker (Bellco Biotechnology, model: 7644-10115 )

  4. Vortexer (Denville Scientific, model: S7030 )

  5. Fume hood

  6. Nitrogen evaporator (Thermo Fisher Scientific, model: TS-18825 )

  7. Oven (Thermo Fisher Scientific, model: 13-247-650G )

  8. Personal safety equipment: lab coat, nitrile gloves, and eye protection

  9. Cork borer

  10. TLC developing tank

  11. Scissors

  12. Survey Meter (Ludlum Measurements, model: Model 3 )

  13. Autoradiography cassette (FUJIFILM, model: BAS 2040 )

  14. Phosphor screen (FUJIFILM, model: BAS-SR-2040 )

  15. Scintillation analyzer (PerkinElmer, model: Tri-Carb 2900TR )

  16. Typhoon FLA 7000 biomolecular imager (GE Healthcare)

  17. -80 °C and -20 °C freezer

  18. Centrifuge (Eppendorf, model: 5425 )

Software

  1. ImageQuant5.2 (GE Healthcare)

Procedure

English
中文翻译

文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Yu, L., Zhou, C., Fan, J. and Xu, C. (2021). Using 14C-acetate Pulse-chase Labeling to Study Fatty Acid and Glycerolipid Metabolism in Plant Leaves. Bio-protocol 11(3): e3900. DOI: 10.21769/BioProtoc.3900.

ris ris Download Citation in RIS Format

分类

生物化学 > 脂质

植物科学 > 植物生物化学 > 代谢物

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

0/150

tip 提问指南

+ 问题描述

写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。

post 发布问题
0 Q&A
pen 提交稿件