How to quantify magic spots - a brief overview of (p)ppGpp detection and quantitation methods

Guanosine tetra- and penta-phosphates, collectively known as (p)ppGpp, are well-known second messengers of cellular stress responses in bacteria and plants. Their intracellular concentration is tightly regulated and can vary widely-from undetectable levels under optimal growth conditions, through intermediate concentrations, to extremely high levels that match or even exceed GTP concentrations when cells are exposed to severe stress. Importantly, the effects exerted by (p)ppGpp are often concentration-dependent, making their quantitative analysis a crucial aspect of studying cellular responses to stress. To gain a deeper understanding of the regulatory mechanisms associated with (p)ppGpp, it is essential to monitor its accumulation in vivo and conduct detailed molecular studies in vitro. Various methods have been developed for detecting and quantifying (p)ppGpp, enabling researchers to track its levels in living cells and analyse its function under controlled laboratory conditions. In this work, we provide an overview of the available techniques for (p)ppGpp detection and quantification. We present their advantages, limitations, and potential applications in research on metabolic regulation and cellular stress responses.