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Differential Radial Capillary Action of Ligand Assay: A High-Throughput Technique to Identify Bacterial Nucleotide Second Messenger-Binding Intracellular Proteins
Overview
This video demonstrates the differential radial capillary action of ligand assay — a technique for systematic high-throughput screening of intracellular nucleotide second messenger-binding proteins. This technique allows for direct binding of the messengers to the overexpressed target protein in the cell lysate — bypassing the need for protein purification.
Protocol
1. Preparation of whole cell lysates
- Inoculate the E. coli K-12 ASKA ORFeome collection strains into 1.5 mL Lysogeny broth (LB) containing 25 µg/mL chloramphenicol in 96-well deep well plates. Grow overnight (O/N) for 18 h at 30 °C with shaking at 160 rpm. On the next day, add isopropyl β-d-1-thiogalactopyranoside (IPTG) (final 0.5 mM) to the O/N cultures to induce protein expression at 30 °C for 6 h.
- Pellet cells at 500 x g for 10 min. Freeze the pellets at -80.......
Representative Results
Figure 1: Quantification and calculation of the binding fraction. See the text for details. Briefly, the DRaCALA spots will be analyzed by drawing two circles that circumscribe the whole spot and the inner dark dot (i.e., the retained (p)ppGpp due to the binding of the tested protein). The specific binding signal is the radioactive signal of the inner circle (S1) after subtr.......
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Source: Schicketanz, M. L., et al. Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA). J. Vis. Exp. (2021)
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