Thin-Layer Chromatography-Based Separation of (p)ppGpp Nucleotides Isolated from Stress-Induced Bacteria

Abstract

Source: Fernández-Coll, L. and Cashel, M. Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography. J. Vis. Exp. (2019)

This video presents a high-throughput protocol for measuring the levels and dynamics of the bacterial stress-response nucleotides ppGpp and pppGpp using thin-layer chromatography (TLC). The method provides a powerful, rapid, and cost-effective approach to quantifying (p)ppGpp in bacterial cultures under various stress conditions, enabling high-resolution analysis of bacterial physiology and the stringent response.

Protocol

1. Thin-layer Chromatography
   1. With a soft pencil, mark an origin line 1 cm from the edge of the 20 cm x 20 cm Polyethylenimine-Cellulose (PEI-cellulose) thin-layer chromatography (TLC) plate that has had 5 cm removed from the top with scissors. Apply 5 µL as a droplet on the PEI surface for each sample. Begin ascending development without allowing this spot to dry, which improves resolution by minimizing streaking.
   2. Do ascending development in a tank with a layer of 1.5 M KH₂PO₄ (pH 3.4) solution shallow enough so that liquid does not touch the origin sample spot. Cover the tank with an air-tight s....

Results

Figure 1: Representative TLC analysis. Schematic representation of the results obtained af.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Autoradiography film	Denville scientific inc.	E3218
Storage phosphor screen	Kodak	So230
TLC PEI Cellulose F	Merk-Millipore	1.05579.0001
Typhoon 9400 imager	GE Healthcare
KH₂PO₄	Fisher Biotech	BP362-500
H₃PO₄	J.T.Baker	0260-02