用[14C]谷氨酸进行体内叶绿素放射性标记并采用薄层色谱分析法（TLC）对标记的叶绿素相对量化测定

Roman  Sobotka; Luděk Kořený; Jana Kopečná; Miroslav  Oborník

doi:10.21769/BioProtoc.249

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Peer-reviewed

Radiolabeling of Chlorophyll by [14C]Glutamic Acid in vivo and Relative Quantification of Labeled Chlorophyll by Using Thin Layer Chromatography (TLC)

用[14C]谷氨酸进行体内叶绿素放射性标记并采用薄层色谱分析法（TLC）对标记的叶绿素相对量化测定

RS Roman Sobotka email

LK Luděk Kořený

JK Jana Kopečná

MO Miroslav Oborník

发布: 2012年08月20日第2卷第16期 DOI: 10.21769/BioProtoc.249 浏览次数: 9457

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Abstract

This is an accurate method to assess the rate of chlorophyll biosynthesis in vivo in cyanobacteria. Given that labeled glutamate is used as the very early precursor of chlorophyll together with a short pulse of labeling (30 min), this method provides information about the metabolic flow through the whole chlorophyll biosynthetic pathway on a short timescale.

Keywords: Chlorophyll biosynthesis (叶绿素的生物合成)

14C radiolabeling (14c标记)

Cyanobacteria (蓝藻)

Synechocystis (集胞藻)

Thin Layer Chromatography (薄层色谱法)

Materials and Reagents

Synechocystis PCC 6803
Glutamic acid [U-C14] (ARC 0165A, American Radiolabeled Chemicals) ([¹⁴C]Glu)
Methanol
25% ammonia solution

1 M NaCl
Hexane
10% KOH
Petroleum ether
Chloroform
1 M Na₂HPO₄
1 M NaH₂PO₄
X-ray film (Eastman Kodak Company)
NH₄OH
1 M TES [2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid]/NaOH (pH 8.2)
Growth medium BG11

Equipment

10 ml Headspace vials (Sigma-Aldrich)
Water bath shaker
2 ml o-ring cap tubes
Glass beads (100-200 μm)
Vortex
Tabletop centrifuge (MiniSpin plus, Eppendorf)
Speedvac Concentrator plus (Eppendorf)
Silica gel TLC plate (SIL G-25, MACHEREY-NAGEL)
Rectangular TLC developing tank (Sigma-Aldrich)
Mikro 22R centrifuge (Hettich)
MiniSpin centrifuge (Eppendorf)

Procedure

English

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Sobotka, R., Kořený, L., Kopečná, J. and Oborník, M. (2012). Radiolabeling of Chlorophyll by [14C]Glutamic Acid in vivo and Relative Quantification of Labeled Chlorophyll by Using Thin Layer Chromatography (TLC). Bio-protocol 2(16): e249. DOI: 10.21769/BioProtoc.249.