Thin-layer chromatography for ppGpp detection.

The method described by Cashel for (p)ppGpp detection from E. coli was adapted as follows (80). A909 WT, ΔrelA, and ΔcodY were grown overnight on LB plates with appropriate selection. Individual colonies were scraped from the agar and resuspended in 5 ml MOPS (morpholinepropanesulfonic acid) minimal medium without supplemental phosphorus or serine to achieve an OD₆₀₀ of 0.9. Each bacterial suspension was then divided into two 65-μl aliquots, to which 10 μl ³²P was added for a final concentration of >100 mCi/ml. For the SR activation conditions, SHX in MOPS was added to a final concentration of 1 mg/ml; control samples were spiked with an equal volume of MOPS without SHX. After 30 min, one volume of 13 M formic acid was added to the samples, which were then subjected to three sequential freeze-thaw cycles. The bacterial debris was pelleted by centrifugation, and the supernatants spotted to polyethyleneimine cellulose-coated thin-layer chromatography plates, where they were allowed to dry. The plates were run in covered beakers with 1.5 M KH₂PO₄. Once this buffer was near the top of the plate, the plate was dried and exposed to autoradiography film overnight, which was then developed and photographed.