Differential Radial Capillary Action of Ligand Assay: A High-Throughput Technique to Identify Bacterial Nucleotide Second Messenger-Binding Intracellular Proteins
Transcript
Bacteria produce nucleotide second messengers, NSMs – intracellular signaling molecules – in response to appropriate stimuli. These molecules bind to target proteins and regulate bacterial functions.
To identify these target proteins using differential radial capillary action of ligand assay, take a multi-well plate with bacterial cell lysate containing soluble proteins. These proteins are derived from different bacterial clones expressing individual ORFs – open reading frames – in the genome, ensuring each well has a distinct protein.
Add a mix of guanosine tetra- and penta-phosphates – NSMs labeled with radioactive phosphorus. These molecules bind to target proteins in the lysate. With a pin tool, collect an equal amount of liquid from each well. Place the tool on a nitrocellulose membrane to deposit the liquid as spots.
Target proteins bind to the membrane through non-covalent interactions preventing their diffusion. This sequesters the bound radiolabeled NSMs at the central point of application. Free NSMs radially diffuse with the liquid phase via capillary action.
Use phosphor imaging to detect the radioactive signals and obtain a digital image of the spots. Define the spots using two circles. The outer one depicts the periphery of the diffused NSMs. The inner circle represents the sequestered NSMs.
Calculate the binding fraction – the intensity of radioactivity detected from the inner circle over the total radioactivity of the diffused spot. Locate spots with high binding fractions to identify the wells with NSM-bound target proteins.
