Figure 2
Isocratic strong anion exchange HPLC resolves ppGpp and pppGpp effectively, but is inefficient at resolving other nucleotide species. (a) Nucleotide standards (0.3 nmol each of ADP, GDP, CTP, ATP, UTP, GTP and 0.6 nmol of ppGpp) were run on isocratic SAX-HPLC using absorbance at 252 nm as a readout. (b) The system was used to resolve the nucleotides from E. coli cells both before and 5 minutes after induction of the stringent response. The stringent response was induced by mupirocin added to a final concentration of 150 µg/ml. Sample preparation is described in the Methods section. A 5 µm SphereClone column 4.6 \(\times \) 150 mm was run with a buffer containing 0.36 M NH4H2PO4 pH 3.4, 2.5% acetonitrile at 26 °C at a flow rate of 1.5 ml/min.
