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

Materials and Reagents

1. Synechocystis PCC 6803
   
2. Glutamic acid [U-C14] (ARC 0165A, American Radiolabeled Chemicals) ([¹⁴C]Glu)
3. Methanol
4. 25% ammonia solution
   
5. 1 M NaCl
6. Hexane
7. 10% KOH
8. Petroleum ether
9. Chloroform
10. 1 M Na₂HPO₄
11. 1 M NaH₂PO₄
12. X-ray film (Eastman Kodak Company)
13. NH₄OH
    
14. 1 M TES [2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid]/NaOH (pH 8.2)
15. Growth medium BG11

Equipment

1. 10 ml Headspace vials (Sigma-Aldrich)
2. Water bath shaker
3. 2 ml o-ring cap tubes
   
4. Glass beads (100-200 μm)
   
5. Vortex
6. Tabletop centrifuge (MiniSpin plus, Eppendorf)
7. Speedvac Concentrator plus (Eppendorf)
8. Silica gel TLC plate (SIL G-25, MACHEREY-NAGEL)
9. Rectangular TLC developing tank (Sigma-Aldrich)
10. Mikro 22R centrifuge (Hettich)
11. MiniSpin centrifuge (Eppendorf)

Procedure